Recombinant host cells comprising phosphoketalase

ABSTRACT

The present invention is related to recombinant host cells comprising: (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The present invention is also related to recombinant host cells further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/335,734, filed on Jul. 18, 2014 which is a continuation of U.S. patent application Ser. No. 13/161,168, filed on Jun. 15, 2011, now U.S. Pat. No. 8,871,488, which is related to and claims the benefit of priority of U.S. Provisional Patent Application No. 61/356,379, filed on Jun. 18, 2010, the entirety of each is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of industrial microbiology. The invention relates to recombinant host cells comprising (i) a modification in an endogenous gene encoding a polypeptide that converts pyruvate to acetyl-CoA, acetaldehyde or acetyl-phosphate and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The invention also relates to recombinant host cells comprising (i) a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase (PDC) activity, or a modification in an endogenous polypeptide having PDC activity, and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. The invention also relates to recombinant host cells further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Additionally, the invention relates to methods of making and using such recombinant host cells including, for example, methods of increasing cell growth, methods of reducing or eliminating the requirement of an exogenous carbon substrate for cell growth, methods of increasing glucose consumption and methods of increasing the production of a product of a pyruvate-utilizing pathway.

BACKGROUND OF THE INVENTION

Global demand for liquid transportation fuel is projected to strain the ability to meet certain environmentally driven goals, for example, the conservation of oil reserves and limitation of greenhouse gas emissions. Such demand has driven the development of technology which allows utilization of renewable resources to mitigate the depletion of oil reserves and to minimize greenhouse gas emissions.

Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly. The production of isobutanol from plant-derived raw materials would minimize greenhouse gas emissions and would represent an advance in the art.

2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). 2,3-butanediol can be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol may be used as plasticizers (Voloch et al., “Fermentation Derived 2,3-Butanediol,” in Comprehensive Biotechnology, Pergamon Press Ltd., England Vol. 2, Section 3:933-947 (1986)).

Microorganisms can be engineered for the expression of biosynthetic pathways that initiate with cellular pyruvate to produce, for example, 2,3-butanediol, 2-butanone, 2-butanol and isobutanol. U.S. Pat. No. 7,851,188 discloses the engineering of recombinant microorganisms for production of isobutanol. U.S. Patent Application Publication Nos. US 20070259410 A1 and US 20070292927 A1 disclose the engineering of recombinant microorganisms for production of 2-butanone or 2-butanol. Multiple pathways are disclosed for biosynthesis of isobutanol and 2-butanol, all of which initiate with cellular pyruvate. Butanediol is an intermediate in the 2-butanol pathway disclosed in U.S. Patent Application Publication No. US 20070292927 A1.

The disruption of the enzyme pyruvate decarboxylase (PDC) in recombinant host cells engineered to express a pyruvate-utilizing biosynthetic pathway has been used to increase the availability of pyruvate for product formation via the biosynthetic pathway. For example, U.S. Application Publication No. US 20070031950 A1 discloses a yeast strain with a disruption of one or more pyruvate decarboxylase genes (a PDC knock-out or PDC-KO) and expression of a D-lactate dehydrogenase gene, which is used for production of D-lactic acid. U.S. Application Publication No. US 20050059136 A1 discloses glucose tolerant two-carbon source-independent (GCSI) yeast strains with no PDC activity, which may have an exogenous lactate dehydrogenase gene. Nevoigt and Stahl (Yeast 12:1331-1337 (1996)) describe the impact of reduced PDC and increased NAD-dependent glycerol-3-phosphate dehydrogenase in Saccharomyces cerevisiae on glycerol yield. U.S. Application Publication No. 20090305363 A1 discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of PDC activity.

While PDC-KO recombinant host cells can be used to produce the products of pyruvate-utilizing biosynthetic pathways, PDC-KO recombinant host cells require exogenous carbon substrate supplementation (e.g., ethanol or acetate) for their growth (Flikweert et al. 1999. FEMS Microbiol. Lett. 174(1):73-79 “Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae”). A similar auxotrophy is observed in Escherichia coli strains carrying a mutation of one or more genes encoding pyruvate dehydrogenase (Langley and Guest, 1977, J. Gen. Microbiol. 99:263-276).

In commercial applications, addition of exogenous carbon substrate in addition to the substrate converted to a desired product can lead to increased costs. There remains a need in the art for recombinant host cells with reduced or eliminated need for exogenous carbon substrate supplementation.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention relates to a recombinant host cell comprising (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate, or acetyl-CoA; and ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to such a recombinant host cell further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In embodiments, the polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate, or acetyl-CoA is pyruvate decarboxylase, pyruvate-formate lyase, pyruvate dehydrogenase, pyruvate oxidase, or pyruvate:ferredoxin oxidoreductase.

One aspect of the invention relates to a recombinant host cell comprising (i) a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity or in an endogenous polypeptide having pyruvate decarboxylase activity; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to such a recombinant host cell further comprising (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

One aspect of the invention relates to a recombinant host cell comprising (i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity; and (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Another aspect of the invention relates to a recombinant host cell further comprising: (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Another aspect of invention relates to a reduced or eliminated requirement of such cells for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii) or (iii). Another aspect of the invention relates to the growth of such host cells in culture media that is not supplemented with an exogenous two-carbon substrate, for example, at a growth rate substantially equivalent to, or greater than, the growth rate of a host cell comprising (i) and not (ii) or (iii) in culture media supplemented with an exogenous two-carbon substrate.

In one aspect of the invention, the recombinant host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, or Saccharomyces. In another aspect of the invention, the recombinant host cell is S. cerevisiae

In another aspect of the invention, the recombinant host cell expresses a pyruvate-utilizing biosynthetic pathway including, for example, a biosynthetic pathway for a product such as 2,3-butanediol, isobutanol, 2-butanol, 2-butanone, valine, leucine, alanine, lactic acid, malic acid, fumaric acid, succinic acid, or isoamyl alcohol. Another aspect of the invention relates to expression of an isobutanol biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to 2,3-dihydroxyisovalerate; (iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (iv) 2-ketoisovalerate to isobutyraldehyde; and (v) isobutyraldehyde to isobutanol. Another aspect of the invention relates to expression of a 2-butanone biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; and (iv) 2,3-butanediol to 2-butanone.

Another aspect of the invention relates to expression of a 2-butanol biosynthetic pathway in the recombinant host cell comprising at least one DNA molecule encoding a polypeptide that catalyzes a substrate to product conversion selected from the group consisting of (i) pyruvate to acetolactate; (ii) acetolactate to acetoin; (iii) acetoin to 2,3-butanediol; (iv) 2,3-butanediol to 2-butanone; and (v) 2-butanone to 2-butanol.

One aspect of the invention relates to methods for the production of a product selected from the group consisting of 2,3-butanediol, isobutanol, 2-butanol, 2-butanone, valine, leucine, alanine, lactic acid, malic acid, fumaric acid, succinic acid and isoamyl alcohol comprising growing the recombinant host cells described herein under conditions wherein the product is produced and optionally recovering the product. Another aspect of the invention relates to methods of producing a recombinant host cell comprising transforming a host cell comprising at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity with (i) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Another aspect of the invention relates to methods of improving the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In embodiments, the methods further comprise growing the recombinant host cell in media containing limited carbon substrate.

Another aspect of the invention relates to methods of reducing the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Another aspect of the invention relates to methods of eliminating the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, comprising (i) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (ii) transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Still another aspect of the invention relates to methods for increasing the activity of the phosphoketolase pathway in a recombinant host cell comprising (i) providing a recombinant host cell of the invention; and (ii) growing the recombinant host cell under conditions whereby the activity of the phosphoketolase pathway in the recombinant host cell is increased.

In another aspect, the recombinant host cells comprise a phosphoketolase that matches the Profile HMM given in Table 6 with an E value of less than 7.5E-242. In another aspect, the phosphoketolase has at least about 40% identity to at least one of SEQ ID NO: 355, 379, 381, 388, 481, 486, 468, or 504. In another aspect, the phosphoketolase has at least about 90% identity to at least one of SEQ ID NO: 355, 379, 381, 388, 481, 486, 468, or 504. In another aspect, the phosphoketolase matches the Profile HMMs given in Tables 6, 7, 8, and 9 with E values of less than 7.5E-242, 1.1E-124, 2.1E-49, 7.8E-37, respectively. In another aspect, the recombinant host cells further comprise a phosphotransacetylase which matches the Profile HMM given in Table 14 with an E value of less than 5E-34. In another aspect, the phosphotransacetylase has at least about 40% identity to SEQ ID NO: 1475, 1472, 1453, 1422, 1277, 1275, 1206, 1200, 1159, or 1129. In another aspect, the phosphotransacetylase has at least about 90% identity to SEQ ID NO: 1475, 1472, 1453, 1422, 1277, 1275, 1206, 1200, 1159, or 1129.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES AND INCORPORATION OF SEQUENCE LISTING AND TABLES

The various embodiments of the invention can be more fully understood from the detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.

FIG. 1 depicts a schematic representation of the phosphoketolase pathway, including the phosphoketolase and phosphotransacetylase enzymes.

FIG. 2 depicts the growth of PDC-KO yeast strains expressing phosphoketolase and phosphotransacetylase without exogenous carbon substrate supplementation.

FIG. 3 depicts the growth of PDC-KO yeast strains expressing phosphoketolase and/or phosphotransacetylase in without exogenous carbon substrate supplementation.

FIG. 4 depicts a phylogenetic tree of phosphate acetyltransferase (PTA) and phosphate butyryltransferase (PTB) sequences. Multiple sequence alignment was performed with Clustal X using default parameters. Phylogenetic tree was deduced using neighbor-joining method and drawn with Mega 4 software. Marked sequences are as follows: (#, Species, GI#) 1, S. enterica, 56412650; 2, E. coli K12, 88192043; 3, V. parvula, 227371784; 4, C. kluyveri, 153954015; 5, C. Acetobutylicum, 15895019; 6, C. thermocellum, 196254011; 7, M. thermophila, 88192043; 8, S. pyogenes, 48425286; 9, B. subtilis, 58176784; 10, L. fermentum, 227514417; 11, L. plantarum, 28377658; 12, L. sanfranciscensis, 11862872;

FIG. 5 is a plasmid map of pRS426::GPD-xpk1+ADH-eutD map which is described herein.

FIG. 6 depicts the Δpdc1::ilvD(Sm) locus of BP913 after integration of a phosphoketolase pathway vector (described herein).

FIG. 7A shows the growth of an isobutanol-producing strain in the absence (no ETOH) and presence (+ETOH) of EtOH and the absence and presence of the phosphoketolase pathway (xpk). ISO1, ISO2 and ISO3 refer to replicates.

FIG. 7B shows the growth of a second subculture of strains from FIG. 7A.

FIG. 8A to FIG. 8SSS depicts Table 6. Table 6 is a table of the Profile Hidden Markov Model (HMM) for phosphoketolases.

FIG. 9A to FIG. 9GG depicts Table 7. Table 7 is a table of the Profile HMM for phosphoketolase domain XFP_N.

FIG. 10A to FIG. 10P depicts Table 8. Table 8 is a table of the Profile HMM for phosphoketolase domain XFP.

FIG. 11A to FIG. 11R depicts Table 9. Table 9 is a table of the Profile HMM for phosphoketolase domain XFP_C.

FIG. 12A to FIG. 12CC depicts Table 14. Table 14 is a table of the Profile HMM for phosphotransacetylases.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The sequence listing provided herewith is herein incorporated by reference and conforms with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and is consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822. The content of the electronically submitted sequence listing Name: 20110615_CL4871USNA_SeqList.txt; Size: 6.67 MB; and Date of Creation/Modification: Jun. 9, 2011/Jun. 15, 2011 is incorporated herein by reference in its entirety.

SEQ ID NOs: 1-20 are sequences of PDC target gene coding regions and proteins.

SEQ ID NOs: 21-638 are phosphoketolase target gene coding regions and proteins.

SEQ ID NOs: 762-1885 are phosphotransacetylase target gene coding regions and proteins.

SEQ ID NOs: 1893-1897 are hybrid promoter sequences.

SEQ ID NOs: 639-642, 644-654, 656-660, 662-701-714, 725-726, 729-740, 742-748, and 750-761 are primers.

SEQ ID NO: 643 is the vector pRS426::GPD-xpk1+ADH1-eutD.

SEQ ID NO: 655 is the TEF1p-kan-TEF1t gene.

SEQ ID NO: 661 is vector pLA54.

SEQ ID NO: 715 is vector pRS423::pGAL1-cre.

SEQ ID NO: 716 is the vector pLH468-sadB.

SEQ ID NOs: 717 and 718 are the amino acid and nucleic acid sequences for sadB from Achromobacter xylosoxidans.

SEQ ID NO: 719 is the kivD coding region from L. lactis.

SEQ ID NO: 720 is the plasmid pRS425::GPM-sadB.

SEQ ID NO: 721 is the GPM promoter.

SEQ ID NO: 722 is the ADH1 terminator.

SEQ ID NO: 723 is the GPM-sadB-ADHt segment.

SEQ ID NO: 724 is the pUC19-URA3 plasmid.

SEQ ID NO: 741 is the ilvD-FBA1t segment.

SEQ ID NO: 749 is URA3r2 template DNA.

SEQ ID NO: 1886 is the ilvD coding region from S. mutans.

SEQ ID NO: 1888 is vector pLH468.

SEQ ID NO: 1898 is pUC19-URA3::pdc1::GPD-xpk1+ADH1-eutD.

SEQ ID NOs: 1899-1906 are the sequences of modified S. cerevisiae loci.

SEQ ID NO: 1907 is the sequence of pLH702.

SEQ ID NO: 1908 is the sequence of pYZ067DkivDDhADH

SEQ ID NO: 1909 is the amino acid sequence of ALD6.

SEQ ID NO: 1910 is the amino acid sequence of K9D3.

SEQ ID NO: 1911 is the amino acid sequence of K9G9.

SEQ ID NO: 1912 is the amino acid sequence of YMR226c.

SEQ ID NOs: 1913 and 1914 are the nucleic acid and amino acid sequences of AFT1.

SEQ ID NOs: 1915 and 1916 are the nucleic acid and amino acid sequences of AFT2.

SEQ ID NOs: 1917 and 1918 are the nucleic acid and amino acid sequences of FRA2.

SEQ ID NOs: 1919 and 1920 are the nucleic acid and amino acid sequences of GRX3.

SEQ ID NOs: 1921 and 1922 are the nucleic acid and amino acid sequences of CCC1.

SEQ ID NO: 1923 is the amino acid sequence of an alcohol dehydrogenase from Beijerinckia indica.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have solved the stated problem by reducing or eliminating the need for providing two substrates, one of which is converted to a desired product, the other fully or partly into acetyl-CoA by recombinant host cells requiring such supplementation for growth comprising the expression of enzymes of the phosphoketolase pathway in such cells. One such enzyme, phosphoketolase (Enzyme Commission Number EC 4.1.2.9), catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl-phosphate (Heath et al., J. Biol. Chem. 231: 1009-29; 1958). Another such enzyme is phosphotransacetylase (Enzyme Commission Number EC 2.3.1.8) which converts acetyl-phosphate into acetyl-CoA.

Applicants have provided PDC-KO recombinant host cells comprising a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity, and optionally a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. Such cells exhibit a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for their growth compared to PDC-KO cells. Applicants have also provided methods of making and using such recombinant host cells including, for example, methods of increasing cell growth, methods of reducing or eliminating the requirement of an exogenous two-carbon substrate for cell growth, methods of increasing glucose consumption and methods of increasing the production of a product of a pyruvate-utilizing pathway.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference, unless only specific sections of patents or patent publications are indicated to be incorporated by reference.

Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, suitable methods and materials are described below. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

In order to further define this invention, the following terms, abbreviations and definitions are provided.

As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

As used herein, the term “about” modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In one embodiment, the term “about” means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term “butanol” as used herein, refers to 2-butanol, 1-butanol, isobutanol, or mixtures thereof.

The term “pyruvate-utilizing biosynthetic pathway” refers to an enzyme pathway to produce a biosynthetic product from pyruvate.

The term “isobutanol biosynthetic pathway” refers to an enzyme pathway to produce isobutanol from pyruvate.

The term “2-butanol biosynthetic pathway” refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term “2-butanone biosynthetic pathway” refers to an enzyme pathway to produce 2-butanone from pyruvate.

The terms “pdc-,” “PDC knock-out,” or “PDC-KO” as used herein refer to a cell that has a genetic modification to inactivate or reduce expression of at least one gene encoding pyruvate decarboxylase (PDC) so that the cell substantially or completely lacks pyruvate decarboxylase enzyme activity. If the cell has more than one expressed (active) PDC gene, then each of the active PDC genes may be inactivated or have minimal expression thereby producing a pdc-cell.

The term “carbon substrate” refers to a carbon source capable of being metabolized by the recombinant host cells disclosed herein. Non-limiting examples of carbon substrates are provided herein and include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, ethanol, lactate, succinate, glycerol, carbon dioxide, methanol, glucose, fructose, sucrose, xylose, arabinose, dextrose, or mixtures thereof.

The term “exogenous two-carbon substrate” refers to the carbon source provided to be metabolized into acetyl-CoA by a host cell that lacks the ability to convert pyruvic acid into acetyl-CoA. The term is used to distinguish from the carbon substrate which is converted into a pyruvate-derived product by a pyruvate-utilizing biosynthetic pathway, herein also referred to as the “pathway substrate” which includes, for example, glucose.

The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to a nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can contain the nucleotide sequence of the full-length cDNA sequence, or a fragment thereof, including the untranslated 5′ and 3′ sequences and the coding sequences. The polynucleotide can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. “Polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

A polynucleotide sequence may be referred to as “isolated,” in which it has been removed from its native environment. For example, a heterologous polynucleotide encoding a polypeptide or polypeptide fragment having dihydroxy-acid dehydratase activity contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. An isolated polynucleotide fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term “gene” refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “heterologous gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. “Heterologous gene” includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

As used herein the term “coding region” refers to a DNA sequence that codes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

As used herein, the term “variant” refers to a polypeptide differing from a specifically recited polypeptide of the invention by amino acid insertions, deletions, mutations, and substitutions, created using, e.g., recombinant DNA techniques, such as mutagenesis. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous polypeptides, e.g., yeast or bacterial, and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequences.

Alternatively, recombinant polynucleotide variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector for expression. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide.

Amino acid “substitutions” may be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they may be the result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, “non-conservative” amino acid substitutions may be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids. “Insertions” or “deletions” may be within the range of variation as structurally or functionally tolerated by the recombinant proteins. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

The term “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. For example, it will be understood that “FBA1 promoter” can be used to refer to a fragment derived from the promoter region of the FBA1 gene.

The term “terminator” as used herein refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The 3′ region can influence the transcription, RNA processing or stability, or translation of the associated coding sequence. It is recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical terminator activity. For example, it will be understood that “CYC1 terminator” can be used to refer to a fragment derived from the terminator region of the CYC1 gene.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

The term “overexpression,” as used herein, refers to expression that is higher than endogenous expression of the same or related gene. A heterologous gene is overexpressed if its expression is higher than that of a comparable endogenous gene.

As used herein the term “transformation” refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid” and “vector” as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

As used herein the term “codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term “codon-optimized” as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.

TABLE 1 The Standard Genetic Code T C A G T TTT Phe(F) TCT Ser (S) TAT Tyr (Y) TGT Cys(C) TTC ″ TCC ″ TAC ″ TGC TTA Leu (L) TCA ″ TAA Stop TGA Stop TTG ″ TCG ″ TAG Stop TGG Trp(W) C CTT Leu(L) CCT Pro (P) CAT His(H) CGT Arg(R) CTC ″ CCC ″ CAC ″ CGC ″ CTA ″ CCA ″ CAA Gln(Q) CGA ″ CTG ″ CCG ″ CAG ″ CGG ″ A ATT Ile(I) ACT Thr(T) ATT Asn(N) AGT Ser(S) ATC ″ ACC ″ AAC ″ AGC ″ ATA ″ ACA ″ AAA Lys(K) AGA Arg(R) ATG Met (M) ACG ″ AAG ″ AGG ″ G GTT Val(V) GCT ALA(A) GAT Asp(D) GGT Gly(G) GTC ″ GCC ″ GAC ″ GGC ″ GTA ″ GCA ″ GAA Glu(E) GGA ″ GTG ″ GCG ″ GAG ″ GGG ″

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference, or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http://www.kazusa.or.jp/codon/(visited Mar. 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 Feb. 2002], are reproduced below as Table 2. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. Table 2 has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

TABLE 2 Codon Usage Table for Saccharomyces cerevisiae Genes Amino Frequency per Acid Codon Number thousand Phe UUU 170666 26.1 Phe UUC 120510 18.4 Leu UUA 170884 26.2 Leu UUG 177573 27.2 Leu CUU 80076 12.3 Leu CUC 35545 5.4 Leu CUA 87619 13.4 Leu CUG 68494 10.5 Ile AUU 196893 30.1 Ile AUC 112176 17.2 Ile AUA 116254 17.8 Met AUG 136805 20.9 Val GUU 144243 22.1 Val GUC 76947 11.8 Val GUA 76927 11.8 Val GUG 70337 10.8 Ser UCU 153557 23.5 Ser UCC 92923 14.2 Ser UCA 122028 18.7 Ser UCG 55951 8.6 Ser AGU 92466 14.2 Ser AGC 63726 9.8 Pro CCU 88263 13.5 Pro CCC 44309 6.8 Pro CCA 119641 18.3 Pro CCG 34597 5.3 Thr ACU 132522 20.3 Thr ACC 83207 12.7 Thr ACA 116084 17.8 Thr ACG 52045 8.0 Ala GCU 138358 21.2 Ala GCC 82357 12.6 Ala GCA 105910 16.2 Ala GCG 40358 6.2 Tyr UAU 122728 18.8 Tyr UAC 96596 14.8 His CAU 89007 13.6 His CAC 50785 7.8 Gln CAA 178251 27.3 Gln CAG 79121 12.1 Asn AAU 233124 35.7 Asn AAC 162199 24.8 Lys AAA 273618 41.9 Lys AAG 201361 30.8 Asp GAU 245641 37.6 Asp GAC 132048 20.2 Glu GAA 297944 45.6 Glu GAG 125717 19.2 Cys UGU 52903 8.1 Cys UGC 31095 4.8 Trp UGG 67789 10.4 Arg CGU 41791 6.4 Arg CGC 16993 2.6 Arg CGA 19562 3.0 Arg CGG 11351 1.7 Arg AGA 139081 21.3 Arg AGG 60289 9.2 Gly GGU 156109 23.9 Gly GGC 63903 9.8 Gly GGA 71216 10.9 Gly GGG 39359 6.0 Stop UAA 6913 1.1 Stop UAG 3312 0.5 Stop UGA 4447 0.7

By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.

Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the “EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, Wis., the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, Md., and the “backtranslate” function in the GCG-Wisconsin Package, available from Accelrys, Inc., San Diego, Calif. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the “backtranslation” function (Entelechon GmbH, Regensburg, Germany) and the “backtranseq” function (NRC Saskatoon Bioinformatics, Saskatoon, Saskatchewan, Canada). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art.

Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as “synthetic gene designer” (University of Maryland, Baltimore, Md.).

A polynucleotide or nucleic acid fragment is “hybridizable” to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringent conditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washes with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the “Clustal method of alignment” which encompasses several varieties of the algorithm including the “Clustal V method of alignment” corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program. Additionally the “Clustal W method of alignment” is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) and found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a “percent identity” by viewing the “sequence distances” table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, such as from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 55% to 100% may be useful in describing the present invention, such as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning. A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods used here are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Other molecular tools and techniques are known in the art and include splicing by overlapping extension polymerase chain reaction (PCR) (Yu, et al. (2004) Fungal Genet. Biol. 41:973-981), positive selection for mutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J. D. et al. (1984) Mol. Gen. Genet. 197, 345-346; M A Romanos, et al. Nucleic Acids Res. 1991 Jan. 11; 19(1): 187), the cre-lox site-specific recombination system as well as mutant lox sites and FLP substrate mutations (Sauer, B. (1987) Mol Cell Biol 7: 2087-2096; Senecoff, et al. (1988) Journal of Molecular Biology, Volume 201, Issue 2, Pages 405-421; Albert, et al. (1995) The Plant Journal. Volume 7, Issue 4, pages 649-659), “seamless” gene deletion (Akada, et al. (2006) Yeast; 23(5):399-405), and gap repair methodology (Ma et al., Genetics 58:201-216; 1981). Applicants have discovered that activation of the phosphoketolase pathway in a recombinant host cell comprising a modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity or a modification in an endogenous polypeptide having pyruvate decarboxylase activity, reduces or eliminates the need for an exogenous carbon substrate for the growth of such a cell. In embodiments, the recombinant host cells comprise (i) at least one deletion, mutation and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity); (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

The genetic manipulations of the host cells described herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In embodiments, the recombinant host cells disclosed herein can be any bacteria, yeast or fungi host useful for genetic modification and recombinant gene expression. In other embodiments, a recombinant host cell can be a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In other embodiments, the host cell can be Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Candida glabrata, Candida albicans, Pichia stipitis, Yarrowia lipolytica, E. coli, or L. plantarum. In still other embodiments, the host cell is a yeast host cell. In some embodiments, the host cell is a member of the genera Saccharomyces. In some embodiments, the host cell is Kluyveromyces lactis, Candida glabrata or Schizosaccharomyces pombe. In some embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

Sources of Acetyl-CoA

Acetyl-CoA is a major cellular building block, required for the synthesis of fatty acids, sterols, and lysine. Pyruvate is often a major contributor to the acetyl-CoA pool. Pyruvate dehydrogenase catalyzes the direct conversion of pyruvate to acetyl-CoA (E.C. 1.2.4.1, E.C. 1.2.1.51) or acetate (E.C. 1.2.2.2) and is almost ubiquitous in nature. Other enzymes involved in conversion of pyruvate to acetyl-CoA, acetyl-phosphate or acetate include pyruvate-formate lyase (E.C. 2.3.1.54), pyruvate oxidase (E.C. 1.2.3.3, E.C. 1.2.3.6), pyruvate-ferredoxin oxidoreductase (E.C. 1.2.7.1), and pyruvate decarboxylase (E.C. 4.1.1.1). Genetic modifications made to a host cell to conserve the pyruvate pool for a product of interest may include those that restrict conversion to acetyl-CoA, leading to decreased growth in the absence of an exogenously supplied two-carbon substrate, a carbon substrate that can be readily converted to acetyl-CoA independent of pyruvate (e.g. ethanol or acetate). An example is the documented auxotrophy observed in pyruvate decarboxylase deficient Saccharomyces cerevisiae (Flikweert et al. 1999, supra). Another example is the documented auxotrophy observed in pyruvate dehydrogenase deficient Escherichia coli when grown aerobically on glucose (Langley and Guest, 1977, J. Gen. Microbiol. 99:2630276).

Modification of Pyruvate Decarboxylase

In embodiments, the recombinant host cells disclosed herein comprise a modification in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase (PDC) or a modification in an endogenous polypeptide having PDC activity. In embodiments, the recombinant host cells disclosed herein can have a modification or disruption of one or more polynucleotides, genes or polypeptides encoding PDC. In embodiments, the recombinant host cell comprises at least one deletion, mutation, and/or substitution in one or more endogenous polynucleotides or genes encoding a polypeptide having PDC activity, or in one or more endogenous polypeptides having PDC activity. Such modifications, disruptions, deletions, mutations, and/or substitutions can result in PDC activity that is reduced or eliminated, resulting in a PDC knock-out (PDC-KO) phenotype.

In embodiments, the endogenous pyruvate decarboxylase activity of the recombinant host cells disclosed herein converts pyruvate to acetaldehyde, which can then be converted to ethanol or to acetyl-CoA via acetate.

In embodiments, the recombinant host cell is Kluyveromyces lactis containing one gene encoding pyruvate decarboxylase, Candida glabrata containing one gene encoding pyruvate decarboxylase, or Schizosaccharomyces pombe containing one gene encoding pyruvate decarboxylase.

In other embodiments, the recombinant host cell is Saccharomyces cerevisiae containing three isozymes of pyruvate decarboxylase encoded by the pdc1, pdc5, and pdc6 genes, as well as a pyruvate decarboxylase regulatory gene, pdc2. In a non-limiting example in S. cerevisiae, the pdc1 and pdc5 genes, or all three genes, are disrupted. In another non-limiting example in S. cerevisiae, pyruvate decarboxylase activity may be reduced by disrupting the pdc2 regulatory gene. In another non-limiting example in S. cerevisiae, polynucleotides or genes encoding pyruvate decarboxylase proteins such as those having about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to pdc1 or pdc5 can be disrupted.

In embodiments, the polypeptide having PDC activity or the polynucleotide or gene encoding a polypeptide having PDC activity is associated with Enzyme Commission Number EC 4.1.1.1. In other embodiments, a PDC gene of the recombinant host cells disclosed herein is not active under the fermentation conditions used, and therefore such a gene would not need to be modified or inactivated.

Examples of recombinant host cells with reduced pyruvate decarboxylase activity due to disruption of pyruvate decarboxylase encoding genes have been reported, such as for Saccharomyces in Flikweert et al. (Yeast (1996) 12:247-257), for Kluyveromyces in Bianchi et al. (Mol. Microbiol. (1996) 19(1):27-36), and disruption of the regulatory gene in Hohmann (Mol. Gen. Genet. (1993) 241:657-666). Saccharomyces strains having no pyruvate decarboxylase activity are available from the ATCC with Accession #200027 and #200028.

Examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, those of the following table.

TABLE 3 SEQ ID NOs of pyruvate decarboxylase (PDC) target gene coding regions and proteins. SEQ ID NO: SEQ ID NO: Description Nucleic acid Amino acid PDC1 pyruvate decarboxylase from Saccharomyces cerevisiae 1 2 PDC5 pyruvate decarboxylase from Saccharomyces cerevisiae 3 4 PDC6 pyruvate decarboxylase from Saccharomyces cerevisiae 5 6 pyruvate decarboxylase from Candida glabrata 7 8 PDC1 pyruvate decarboxylase from Pichia stipitis 9 10 PDC2 pyruvate decarboxylase from Pichia stipitis 11 12 pyruvate decarboxylase from Kluyveromyces lactis 13 14 pyruvate decarboxylase from Yarrowia lipolytica 15 16 pyruvate decarboxylase from Schizosaccharomyces pombe 17 18 pyruvate decarboxylase from Zygosaccharomyces rouxii 18 20

Other examples of PDC polynucleotides, genes and polypeptides that can be targeted for modification or inactivation in the recombinant host cells disclosed herein include, but are not limited to, PDC polynucleotides, genes and/or polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of the sequences of Table 3.

In embodiments, the sequences of other PDC polynucleotides, genes and/or polypeptides can be identified in the literature and in bioinformatics databases well known to the skilled person using sequences disclosed herein and available in the art. For example, such sequences can be identified through BLAST (as described above) searching of publicly available databases with known PDC encoding polynucleotide or polypeptide sequences. In such a method, identities can be based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, the PDC polynucleotide or polypeptide sequences described herein or known the art can be used to identify other PDC homologs in nature. For example, each of the PDC encoding nucleic acid fragments described herein can be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to (1) methods of nucleic acid hybridization; (2) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and (3) methods of library construction and screening by complementation.

In embodiments, PDC polynucleotides, genes and/or polypeptides related to the recombinant host cells described herein can be modified or disrupted. Many methods for genetic modification and disruption of target genes to reduce or eliminate expression are known to one of ordinary skill in the art and can be used to create the recombinant host cells described herein. Modifications that can be used include, but are not limited to, deletion of the entire gene or a portion of the gene encoding a PDC protein, inserting a DNA fragment into the encoding gene (in either the promoter or coding region) so that the protein is not expressed or expressed at lower levels, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or a less active protein is expressed. In other embodiments, expression of a target gene can be blocked by expression of an antisense RNA or an interfering RNA, and constructs can be introduced that result in cosuppression. In other embodiments, the synthesis or stability of the transcript can be lessened by mutation. In embodiments, the efficiency by which a protein is translated from mRNA can be modulated by mutation. All of these methods can be readily practiced by one skilled in the art making use of the known or identified sequences encoding target proteins.

In other embodiments, DNA sequences surrounding a target PDC coding sequence are also useful in some modification procedures and are available, for example, for yeasts such as Saccharomyces cerevisiae in the complete genome sequence coordinated by Genome Project ID9518 of Genome Projects coordinated by NCBI (National Center for Biotechnology Information) with identifying GOPID #13838. An additional non-limiting example of yeast genomic sequences is that of Candida albicans, which is included in GPID #10771, #10701 and #16373. Other yeast genomic sequences can be readily found by one of skill in the art in publicly available databases.

In other embodiments, DNA sequences surrounding a target PDC coding sequence can be useful for modification methods using homologous recombination. In a non-limiting example of this method, PDC gene flanking sequences can be placed bounding a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the PDC gene. In another non-limiting example, partial PDC gene sequences and PDC gene flanking sequences bounding a selectable marker gene can be used to mediate homologous recombination whereby the marker gene replaces a portion of the target PDC gene. In embodiments, the selectable marker can be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the PDC gene without reactivating the latter. In embodiments, the site-specific recombination leaves behind a recombination site which disrupts expression of the PDC protein. In other embodiments, the homologous recombination vector can be constructed to also leave a deletion in the PDC gene following excision of the selectable marker, as is well known to one skilled in the art.

In other embodiments, deletions can be made to a PDC target gene using mitotic recombination as described in Wach et al. (Yeast, 10:1793-1808; 1994). Such a method can involve preparing a DNA fragment that contains a selectable marker between genomic regions that can be as short as 20 bp, and which bound a target DNA sequence. In other embodiments, this DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome. In embodiments, the linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence ((as described, for example, in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.)).

Moreover, promoter replacement methods can be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh et al. ((2004) Cell 118(1):31-44).

In other embodiments, the PDC target gene encoded activity can be disrupted using random mutagenesis, which can then be followed by screening to identify strains with dependency on carbon substrates for growth. In this type of method, the DNA sequence of the target gene encoding region, or any other region of the genome affecting carbon substrate dependency for growth, need not be known. In embodiments, a screen for cells with reduced PDC activity and/or two-carbon substrate dependency, or other mutants having reduced PDC activity and a reduced or eliminated dependency for exogenous two-carbon substrate for growth, can be useful as recombinant host cells of the invention.

Methods for creating genetic mutations are common and well known in the art and can be applied to the exercise of creating mutants. Commonly used random genetic modification methods (reviewed in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) include spontaneous mutagenesis, mutagenesis caused by mutator genes, chemical mutagenesis, irradiation with UV or X-rays, or transposon mutagenesis.

Chemical mutagenesis of host cells can involve, but is not limited to, treatment with one of the following DNA mutagens: ethyl methanesulfonate (EMS), nitrous acid, diethyl sulfate, or N-methyl-N′-nitro-N-nitroso-guanidine (MNNG). Such methods of mutagenesis have been reviewed in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, chemical mutagenesis with EMS can be performed as described in Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Irradiation with ultraviolet (UV) light or X-rays can also be used to produce random mutagenesis in yeast cells. The primary effect of mutagenesis by UV irradiation is the formation of pyrimidine dimers which disrupt the fidelity of DNA replication. Protocols for UV-mutagenesis of yeast can be found in Spencer et al. (Mutagenesis in Yeast, 1996, Yeast Protocols: Methods in Cell and Molecular Biology. Humana Press, Totowa, N.J.). In embodiments, the introduction of a mutator phenotype can also be used to generate random chromosomal mutations in host cells. In embodiments, common mutator phenotypes can be obtained through disruption of one or more of the following genes: PMS1, MAG1, RAD18 or RAD51. In other embodiments, restoration of the non-mutator phenotype can be obtained by insertion of the wildtype allele. In other embodiments, collections of modified cells produced from any of these or other known random mutagenesis processes may be screened for reduced or eliminated PDC activity.

Genomes have been completely sequenced and annotated and are publicly available for the following yeast strains: Ashbya gossypii ATCC 10895, Candida glabrata CBS 138, Kluyveromyces lactis NRRL Y-1140, Pichia stipitis CBS 6054, Saccharomyces cerevisiae S288c, Schizosaccharomyces pombe 972h-, and Yarrowia lipolytica CLIB122. Typically BLAST (described above) searching of publicly available databases with known PDC polynucleotide or polypeptide sequences, such as those provided herein, is used to identify PDC-encoding sequences of other host cells, such as yeast cells.

Accordingly, it is within the scope of the invention to provide pyruvate decarboxylase polynucleotides and polypeptides having at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any of the PDC polypeptides or polypeptides disclosed herein (SEQ ID NOs: 1-20). Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

The modification of PDC in the host cells disclosed herein to reduce or eliminate PDC activity can be confirmed using methods known in the art. For example, PCR methods well known in the art can be used to confirm deletion of PDC. Other suitable methods will be known to those of skill in the art and include, but are not limited to, lack of growth on yeast extract peptone-dextrose medium (YPD).

Introduction of the Phosphoketolase Pathway

Applicants have found that expression of enzymes associated with the phosphoketolase pathway (e.g., phosphoketolase and/or phosphotransacetylase) results in a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for growth of PDC-KO cells. Phosphoketolases and/or phosphotransacetylases identified as described herein, can be expressed in such cells using methods described herein.

Enzymes of the phosphoketolase pathway include phosphoketolase and phosphotransacetylase (FIG. 1). Phosphoketolase (Enzyme Commission Number EC 4.1.2.9) catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde 3-phosphate and acetyl-phosphate (Heath et al., J. Biol. Chem. 231: 1009-29; 1958). Phosphoketolase activity has been identified in several yeast strains growing with xylose as the sole carbon source but not in yeast strains grown with glucose (Evans and Ratledge, Arch. Microbiol. 139: 48-52; 1984). Inhibitors of phosphoketolase include, but are not limited to, erythrose 4-phosphate and glyceraldehyde 3-phosphate. Phosphotransacetylase (Enzyme Commission Number EC 2.3.1.8) converts acetyl-phosphate into acetyl-CoA.

In embodiments, the phosphoketolase pathway is activated in the recombinant host cells disclosed herein by engineering the cells to express polynucleotides and/or polypeptides encoding phosphoketolase and, optionally, phosphotransacetylase. In embodiments, the recombinant host cells disclosed herein comprise a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the recombinant host cells disclosed herein comprise a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity and a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In other embodiments, the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is overexpressed, or expressed at a level that is higher than endogenous expression of the same or related endogenous gene, if any. In still other embodiments, the heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is overexpressed, or expressed at a level that is higher than endogenous expression of the same or related endogenous gene, if any.

In embodiments, a polypeptide having phosphoketolase activity catalyzes the conversion of xylulose 5-phosphate into glyceraldehyde-3-phosphate and acetyl-phosphate and/or the conversion of fructose-6-phosphate into erythrose-4-phosphate and acetyl-phosphate. In embodiments, the activity of a polypeptide having phosphoketolase activity is inhibited by erythrose 4-phosphate and/or glyceraldehyde 3-phosphate. In other embodiments, a polypeptide having phosphotransacetylase activity catalyzes the conversion of acetyl-phosphate into acetyl-CoA.

Numerous examples of polynucleotides, genes and polypeptides encoding phosphoketolase activity are known in the art and can be used in the recombinant host cells disclosed herein. In embodiments, such a polynucleotide, gene and/or polypeptide can be the xylulose 5-phosphateketolase (XpkA) of Lactobacillus pentosus MD363 (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002). XpkA is the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and exhibits a specific activity of 4.455 μmol/min/mg (Posthuma et al., Appl. Environ. Microbiol. 68: 831-7; 2002). In other embodiments, such a polynucleotide, gene and/or polypeptide can be the phosphoketolase of Leuconostoc mesenteroides which exhibits a specific activity of 9.9 μmol/min/mg and is stable at pH above 4.5 (Goldberg et al., Methods Enzymol. 9: 515-520; 1966). This phosphoketolase exhibits a Km of 4.7 mM for D-xylulose 5-phosphate and a Km of 29 mM for fructose 6-phosphate (Goldberg et al., Methods Enzymol. 9: 515-520; 1966). In other embodiments, such a polynucleotide, gene and/or polypeptide can be the D-xylulose 5-phosphate/D-fructose 6-phosphate phosphoketolase gene xfp from B. lactis, as described, for example, in a pentose-metabolizing S. cerevisiae strain by Sonderegger et al. (Appl. Environ. Microbiol. 70: 2892-7; 2004).

In embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase corresponds to the Enzyme Commission Number EC 4.1.2.9.

In embodiments, host cells comprise a polypeptide having at least about 80%, at least about 85%, at least about 90%, or 100% identity to a polypeptide of Table 4 or an active fragment thereof or a polynucleotide encoding such a polypeptide. In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can include, but is not limited to, a sequence provided in the following tables 4 or 5.

TABLE 4 SEQ ID NOs of phosphoketolase target gene coding regions and proteins SEQ ID SEQ ID NO: NO: Nucleic Amino Description acid acid Amino Acid sequence Xpk1  172  481 MTTDYSSPAYLQKVDKYWRAANYLSVGQLYLKDNPLLQRPL phosphoketolase KASDVKVHPIGHWGTIAGQ from NFIYAHLNRVINKYGLKMFYVEGPGHGGQVMVSNSYLDGTY Lactobacillus TDIYPEITQDVEGMQKLFK plantarum QFSFPGGVASHAAPETPGSIHEGGELGYSISHGVGAILDNP DEIAAVVVGDGESETGPLA TSWQSTKFINPINDGAVLPILNLNGFKISNPTIFGRTSDAK IKEYFESMNWEPIFVEGDD PEKVHPALAKAMDEAVEKIKAIQKHARENNDATLPVWPMIV FRAPKGWTGPKSWDGDKIE GSFRAHQIPIPVDQNDMEHADALVDWLESYQPKELFNEDGS LKDDIKEIIPTGDSRMAAN PITNGGVDPKALNLPNFRDYAVDTSKEGANVKQDMIVWSDY LRDVIKKNPDNFRLFGPDE TMSNRLYGVFETTNRQWMEDIHPDSDQYEAPAGRVLDAQLS EHQAEGWLEGYVLTGRHGL FASYEAFLRVVDSMLTQHFKWLRKANELDWRKKYPSLNIIA ASTVFQQDHNGYTHQDPGA LTHLAEKKPEYIREYLPADANTLLAVGDVIFRSQEKINYVV TSKHPRQQWFSIEEAKQLV DNGLGIIDWASTDQGSEPDIVFAAAGTEPTLETLAAIQLLH DSFPEMKIRFVNVVDILKL RSPEKDPRGLSDAEFDHYFTKDKPVVFAFHGYEDLVRDIFF DRHNHNLYVHGYRENGDIT TPFDVRVMNQMDRFDLAKSAIAAQPAMENTGAAFVQSMDNM LAKHNAYIRDAGTDLPEVN DWQWKGLK XpkA 1890 1889 MSTDYSSPAYLQKVDKYWRAANYLSVGQLYLKDNPLLQRPL phosphoketolase KASDVKVHPIGHWGTIAGQ from NFIYAHLNRVINKYGLKMFYVEGPGHGGQVMVSNSYLDGTY Lactobacillus TDIYPEITQDVEGMQKLFK pentosus QFSFPGGVASHAAPETPGSIHEGGELGYSISHGVGAILDNP MD363 DEIAAVVVGDGESETGPLA TSWQSTKFINPINDGAVLPILNLNGFKISNPTIFGRTSDEK IKQYFESMNWEPIFVEGDD PEKVHPALAKAMDEAVEKIKAIQKNARENDDATLPVWPMIV FRAPKGWTGPKSWDGDKIE GSFRAHQIP1PVDQTDMEHADALVDWLESYQPKELFNEDGS LKDDIKEIIPTGDARMAAN PITNGGVDPKALNLPNFRDYAVDTSKHGANVKQDMIVWSDY LRDVIKKNPDNFRLFGPDE TMSNRLYGVFETTNRQWMEDIHPDSDQYEAPAGRVLDAQLS EHQAEGWLEGYVLTGRHGL FASYEAFLRVVDSMLTQHFKWLRKANELDWRKKYPSLNIIA ASTVFQQDHNGYTHQDPGA LTHLAEKKPEYIREYLPADANSLLAVGDVIFRSQEKINYVV TSKHPRQQWFSIEEAKQLV DNGLGIIDWASTDQGSEPDIVFAAAGTEPTLETLAAIQLLH DSFPDMKIRFVNVVDILKL RSPEKDPRGLSDAEFDHYFTKDKPVVFAFHGYEDLVRDIFF DRHNHNLHVHGYRENGDIT TPFDVRVMNQMDRFDLAKSAIAAQPAMENTGAAFVQDMDNM LAKHNAYIRDAGTDLPEVN DWQWKGLK Xpf D-xylulose   79  388 MTNPVIGTPWQKLDRPVSEEAIEGMDKYWRVANYMSIGQIY 5-phosphate/D- LRSNPLMKEPFTRDDVKHR fructose 6- LVGHWGTTPGLNFLLAHINRLIADHQQNTVFIMGPGHGGPA phosphate GTAQSYIDGTYTEYYPNIT phosphoketolase KDEAGLQKFFRQFSYPGGIPSHFAPETPGSIHEGGELGYAL from B. lactis SHAYGAIMDNPSLFVPCII GDGEAETGPLATGWQSNKLVNPRTDGIVLPILHLNGYKIAN PTILARISDEELHDFFRGM GYHPYEFVAGFDNEDHLSIHRRFAELFETIFDEICDIKAAA QTDDMTRPFYPMLIFRTPK GWTCPKFIDGKKTEGSWRAHQVPLASARDTEAHFEVLKGWM ESYKPEELFNADGSIKEDV TAFMPKGELRIGANPNANGGRIREDLKLPELDQYEITGVKE YGHGWGQVEAPRSLGAYCR DIIKNNPDSFRVFGPDETASNRLNATYEVTKKQWDNGYLSA LVDENMAVTGQVVEQLSEH QCEGFLEAYLLTGRHGIWSSYESFVHVIDSMLNQHAKWLEA TVREIPWRKPISSVNLLVS SHVWRQDHNGFSHQDPGVTSVLLNKTFNNDHVTNIYFATDA NMLLAIAEKCFKSTNKINA IFAGKQPAATWITLDEARAELEAGAAEWKWASNAKSNDEVQ VVLAAAGDVPTQEIMAASD ALNKMGIKFKVVNVVDLIKLQSSKENDEAMSDEDFADLFTA DKPVLFAYHSYAQDVRGLI YDRPNHDNFTVVGYKEQGSTTTPFDMVRVNDMDRYALQAKA LELIDADKYADKINELNEF RKTAFQFAVDNGYDIPEFTDWVYPDVKVDETSMLSATAATA GDNE

In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can have at least about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to that of any one of the sequences of Table 4, wherein the polynucleotide, gene and/or polypeptide encodes a polypeptide having phosphoketolase activity.

In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphoketolase can be used to identify other phosphoketolase polynucleotide, gene and/or polypeptide sequences or to identify phosphoketolase homologs in other cells, as described above for PDC. Such phosphoketolase encoding sequences can be identified, for example, in the literature and/or in bioinformatics databases well known to the skilled person. For example, the identification of phosphoketolase encoding sequences in other cell types using bioinformatics can be accomplished through BLAST (as described above) searching of publicly available databases with known phosphoketolase encoding DNA and polypeptide sequences, such as those provided herein. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additional phosphoketolase target gene coding regions were identified using diversity search, clustering, experimentally verified xylulose-5-phosphate/fructose-6-phosphate phosphoketolases and domain architecture. Briefly, a BLAST search with the experimentally verified sequences with an Evalue cut-off of 0.01 resulted in 595 sequence matches. Clustering with the CD-HIT program at 95% sequence identity and 90% length overlap reduced the number to 436. CD-HIT is a program for clustering large protein database at high sequence identity threshold. The program removes redundant sequences and generates a database of only the representatives. (Clustering of highly homologous sequences to reduce the size of large protein database, Weizhong Li, Lukasz Jaroszewski & Adam Godzik Bioinformatics, (2001) 17:282-283)

Xylulose-5-phosphate/fructose-6-phosphate phosphoketolases have three Pfam domains: XFP_N; XFP; XFP_C. Although each of these domains may be present in several domain architectures, e.g. XFP_N is found in eight architectures. The architecture of interest was determined to be XFP_N; XFP; XFP_C. The cumulative length of the three domains is 760 amino acids.

A structure/function characterization of the phosphoketolases was performed using the HMMER software package. The following information based on the HMMER software user guide gives some description of the way that the hmmbuild program prepares a Profile HMM. A Profile HMM is capable of modeling gapped alignments, e.g. including insertions and deletions, which lets the software describe a complete conserved domain (rather than just a small ungapped motif). Insertions and deletions are modeled using insertion (I) states and deletion (D) states. All columns that contain more than a certain fraction x of gap characters will be assigned as an insert column. By default, x is set to 0.5. Each match state has an I and a D state associated with it. HMMER calls a group of three states (M/D/I) at the same consensus position in the alignment a “node”. These states are interconnected with arrows called state transition probabilities. M and I states are emitters, while D states are silent. The transitions are arranged so that at each node, either the M state is used (and a residue is aligned and scored) or the D state is used (and no residue is aligned, resulting in a deletion-gap character, ‘-’). Insertions occur between nodes, and I states have a self-transition, allowing one or more inserted residues to occur between consensus columns.

The scores of residues in a match state (i.e. match state emission scores), or in an insert state (i.e. insert state emission scores) are proportional to Log_2 (p_x)/(null_x). Where p_x is the probability of an amino acid residue, at a particular position in the alignment, according to the Profile HMM and null_x is the probability according to the Null model. The Null model is a simple one state probabilistic model with pre-calculated set of emission probabilities for each of the 20 amino acids derived from the distribution of amino acids in the SWISSPROT release 24. State transition scores are also calculated as log odds parameters and are proportional to Log_2 (t_x). Where t_x is the probability of transiting to an emitter or non-emitter state.

Using a multiple sequence alignment of experimentally verified sequences containing the architecture of interest XFP_N; XFP; XFP_C, a profile Hidden Markov Model (HMM) was created for representing members of the xylulose-5-phosphate/fructose-6-phosphate phosphoketolases (XPK-XFP). As stated in the user guide, Profile HMMs are statistical models of multiple sequence alignments. They capture position-specific information about how conserved each column of the alignment is, and which amino acid residues are most likely to occur at each position. Thus HMMs have a formal probabilistic basis. Profile HMMs for a large number of protein families are publicly available in the PFAM database (Janelia Farm Research Campus, Ashburn, Va.

Eight xylulose-5-phosphate/fructose-6-phosphate phosphoketolases sequences with experimentally verified function were identified in the BRENDA database:

-   -   1. CBF76492.1 from Aspergillus nidulans FGSC A4 (SEQ ID NO: 355)     -   2. AAR98787.1 from Bifidobacterium longum (SEQ ID NO: 379)     -   3. ZP_03646196.1 from Bifidobacterium bifidum NCIMB 41171 (SEQ         ID NO: 381)     -   4. ZP_02962870.1 from Bifidobacterium animalis subsp. lactis         HN019 (SEQ ID NO: 388)     -   5. NP_786060.1 from Lactobacillus plantarum WCFS1 (SEQ ID NO:         481)     -   6. ZP_03940142.1 from Lactobacillus brevis subsp. gravesensis         ATCC 27305 (SEQ ID NO: 486)     -   7. ZP_03073172.1 from Lactobacillus reuteri 100-23 (SEQ ID NO         468)     -   8. YP_818922.1 from Leuconostoc mesenteroides subsp.         mesenteroides ATCC 8293 (SEQ ID NO: 504)         The BRENDA database is a freely available information system         containing biochemical and molecular information on all         classified enzymes as well as software tools for querying the         database and calculating molecular properties. The database         covers information on classification and nomenclature, reaction         and specificity, functional parameters, occurrence, enzyme         structure and stability, mutants and enzyme engineering,         preparation and isolation, the application of enzymes, and         ligand-related data. (BRENDA, AMENDA and FRENDA the enzyme         information system: new content and tools in 2009. Nucleic Acids         Res. 2009 January; 37 (Database issue):D588-92. Epub 2008         Nov. 4. Chang A, Scheer M, Grote A, Schomburg I, Schomburg D.)         The eight sequences were used to build a profile HMM which is         provided herein as Table 6.

To further identify the proteins of interest, the 436 sequences were searched with four profile HMMs: the generated XPK_XFP_HMM profile HMM provided in Table 6 as well as the three published profiles for the three domains XFP_N; XFP; XFP_C (PFAM DATABASE) described in Tables 7, 8, and 9, respectively. 309 protein sequences which lengths were between 650 amino acids and 900 amino acids, and contained the three domains were retained.

All 309 sequences are at least 40% identical to an experimentally verified phosphoketolase, with exception of 12 sequences that are within 35% identity distance. However, all 309 sequences have a highly significant match to all 4 profile HMMs. The least significant matches have Evalues of 7.5E-242, 1.1E-124, 2.1E-49, 7.8E-37 to XFP_XPK HMM, XFP_N, XFP, and XFP_C profile HMMs respectively. The 309 sequences are provided in Table 5, however, it is understood that any xylulose-5-phosphate/fructose-6-phosphate phosphoketolase identifiable by the method described may be expressed in host cells as described herein. Where accession information is given as “complement(NN_NNNNN.N:X . . . Y)”, it should be understood to mean the reverse complement of nucleotides X to Y of the sequence with Accession number NN_NNNNN.N. Where accession information is given as “join (NNNNNN.N:X . . . Y, NNNNNN.N:Z . . . Q)”, it should be understood to mean the sequence resulting from joining nucleotides X to Y of NNNNNN.N to nucleotides Z to Q of NNNNNN.N.

TABLE 5 SEQ ID NOs of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase target gene coding regions and proteins. GENBANK Amino Acid GENBANK Nucleic Acid Sequence Amino Acid GI Number Nucleotide Sequence Accession Information SEQ ID NO: Accession No. SEQ ID NO: Source Organism 162147402 complement(NC_010125.1: 21 YP_001601863.1 330 Gluconacetobacter 1624982 . . . 1627414) diazotrophicus PAl5 127512024 complement(NC_009092.1: 22 YP_001093221.1 331 Shewanella loihica 1256548 . . . 1258914) PV-4 119774052 complement(NC_008700.1: 23 YP_926792.1 332 Shewanella 1105589 . . . 1107955) amazonensis SB2B 113971300 NC_008321.1: 3541276 . . . 3543642 24 YP_735093.1 333 Shewanella sp. MR-4 126173290 complement(NC_009052.1: 25 YP_001049439.1 334 Shewanella baltica 1204351 . . . 1206717) OS155 163750647 complement(NZ_ABIC01000018.1: 26 ZP_02157884.1 335 Shewanella benthica 47875 . . . 50253) KT99 157374325 complement(NC_009831.1: 27 YP_001472925.1 336 Shewanella 1410491 . . . 1412857) sediminis HAW- EB3 170725643 complement(NC_010506.1: 28 YP_001759669.1 337 Shewanella woodyi 1624098 . . . 1626464) ATCC 51908 167623058 complement(NC_010334.1: 29 YP_001673352.1 338 Shewanella 1364332 . . . 1366698) halifaxensis HAW- EB4 91794082 NC_007954.1: 3270590 . . . 3272956 30 YP_563733.1 339 Shewanella denitrificans OS217 254498997 NZ_ACUL01000224.1: 31 ZP_05111697.1 340 Legionella 8243 . . . 10492 drancourtii LLAP12 239607320 join(EQ999973.1: 7995808 . . . 7995885, 32 EEQ84307.1 341 Ajellomyces EQ999973.1: 7996059 . . . 7996116, dermatitidis ER-3 EQ999973.1: 7996178 . . . 7996412, EQ999973.1: 7996486 . . . 7996638, EQ999973.1: 7996791 . . . 7997016, EQ999973.1: 7997082 . . . 7997548, EQ999973.1: 7997603 . . . 7997705, EQ999973.1: 7997779 . . . 7998604, EQ999973.1: 7998677 . . . 7998717) 261200667 XM_002626688.1: 33 XP_002626734.1 342 Ajellomyces 1 . . . 2034 dermatitidis SLH14081 154276328 XM_001538959.1: 34 XP_001539009.1 343 Ajellomyces 1 . . . 2421 capsulatus NAm1 225555843 join(GG663374.1: 72330 . . . 72492, 35 EEH04133.1 344 Ajellomyces GG663374.1: 72695 . . . 72929, capsulatus G186AR GG663374.1: 73005 . . . 73157, GG663374.1: 73216 . . . 74203, GG663374.1: 74274 . . . 75146) 225681974 join(DS544805.1: 2187102 . . . 2187264, 36 EEH20258.1 345 Paracoccidioides DS544805.1: 2187353 . . . 2187427, brasiliensis Pb03 DS544805.1: 2187521 . . . 2187755, DS544805.1: 2187839 . . . 2187991, DS544805.1: 2188086 . . . 2188395, DS544805.1: 2188462 . . . 2189082, DS544805.1: 2189147 . . . 2189263, DS544805.1: 2189358 . . . 2189973, DS544805.1: 2190044 . . . 2190084) 226289140 join(DS572750.1: 3105630 . . . 3105792, 37 EEH44652.1 346 Paracoccidioides DS572750.1: 3106049 . . . 3106283, brasiliensis Pb18 DS572750.1: 3106367 . . . 3106519, DS572750.1: 3106614 . . . 3106923, DS572750.1: 3107023 . . . 3107610, DS572750.1: 3107675 . . . 3108500, DS572750.1: 3108572 . . . 3108612) 258564014 XM_002582706.1: 38 XP_002582752.1 347 Uncinocarpus reesii 1 . . . 2421 1704 240108203 join(ACFW01000030.1: 39 EER26377.1 348 Coccidioides 1281918 . . . 1282080, posadasii C735 delta ACFW01000030.1: SOWgp 1282133 . . . 1282207, ACFW01000030.1: 1282266 . . . 1282500, ACFW01000030.1: 1282551 . . . 1282703, ACFW01000030.1: 1282757 . . . 1283066, ACFW01000030.1: 1283132 . . . 1283752, ACFW01000030.1: 1283828 . . . 1284653, ACFW01000030.1: 1284726 . . . 1284763) 238838423 join(DS995701.1: 2675053 . . . 2675215, 40 EEQ28085.1 349 Microsporum canis DS995701.1: 2675278 . . . 2675352, CBS 113480 DS995701.1: 2675424 . . . 2675658, DS995701.1: 2675745 . . . 2675897, DS995701.1: 2675972 . . . 2676281, DS995701.1: 2676341 . . . 2676961, DS995701.1: 2677062 . . . 2677836, DS995701.1: 2677864 . . . 2677933, DS995701.1: 2677998 . . . 2678013) 169770631 XM_001819733.1: 41 XP_001819785.1 350 Aspergillus oryzae 1 . . . 2457 RIB40 145232813 XM_001399743.1: 42 XP_001399780.1 351 Aspergillus niger 1 . . . 2421 119491775 XM_001263381.1: 43 XP_001263382.1 352 Neosartorya fischeri 1 . . . 2421 NRRL 181 121705634 XM_001271079.1: 44 XP_001271080.1 353 Aspergillus clavatus 1 . . . 2421 NRRL 1 115396290 XM_001213784.1: 45 XP_001213784.1 354 Aspergillus terreus 1 . . . 2400 NIH2624 259482219 join(BN001303.1: 576345 . . . 576507, 46 CBF76492.1 355 Aspergillus nidulans BN001303.1: 576696 . . . 576930, FGSC A4 BN001303.1: 576981 . . . 577133, BN001303.1: 577185 . . . 577494, BN001303.1: 577544 . . . 578161, BN001303.1: 578210 . . . 579035, BN001303.1: 579091 . . . 579128) 255942289 XM_002561867.1: 47 XP_002561913.1 356 Penicillium 1 . . . 2469 chrysogenum Wisconsin 54-1255 242784458 XM_002480346.1: 48 XP_002480391.1 357 Talaromyces 69 . . . 2489 stipitatus ATCC 10500 212527714 XM_002143978.1: 49 XP_002144014.1 358 Penicillium 139 . . . 2559 marneffei ATCC 18224 70999652 XM_749450.1: 1 . . . 2145 50 XP_754543.1 359 Aspergillus fumigatus Af293 154314622 XM_001556585.1: 51 XP_001556635.1 360 Botryotinia 1 . . . 2061 fuckeliana B05.10 156053245 XM_001592499.1: 52 XP_001592549.1 361 Sclerotinia 1 . . . 2430 sclerotiorum 1980 46124351 XM_386729.1: 1 . . . 2418 53 XP_386729.1 362 Gibberella zeae PH-1 256733824 complement(join(GG698897.1: 54 EEU47171.1 363 Nectria 220636 . . . 220670, haematococca mpVI GG698897.1: 220723 . . . 221650, 77-13-4 GG698897.1: 221704 . . . 222510, GG698897.1: 222576 . . . 222732, GG698897.1: 222783 . . . 223020, GG698897.1: 223072 . . . 223146, GG698897.1: 223199 . . . 223361)) 261354209 join(DS985216.1: 747889 . . . 748126, 55 EEY16637.1 364 Verticillium albo- DS985216.1: 748174 . . . 748564, atrum VaMs.102 DS985216.1: 748620 . . . 748929, DS985216.1: 748985 . . . 749555, DS985216.1: 749607 . . . 749833, DS985216.1: 749946 . . . 750572) 85081035 XM_951556.2: 215 . . . 2662 56 XP_956649.1 365 Neurospora crassa OR74A 145609083 XM_364271.2: 1 . . . 2442 57 XP_364271.2 366 Magnaporthe grisea 70-15 171679277 XM_001904550.1: 58 XP_001904585.1 367 Podospora anserine 1 . . . 2316 169859036 XM_001836107.1: 59 XP_001836159.1 368 Coprinopsis cinerea 1 . . . 2418 okayama7#130 19112755 NM_001021872.1: 60 NP_595963.1 369 Schizosaccharomyces 1 . . . 2478 pombe 213405339 XM_002173405.1: 61 XP_002173441.1 370 Schizosaccharomyces 1 . . . 2469 japonicus yFS275 58267408 XM_570860.1: 39 . . . 2594 62 XP_570860.1 371 Cryptococcus neoformans var. neoformans JEC21 71018661 XM_754468.1: 1 . . . 2682 63 XP_759561.1 372 Ustilago maydis 521 254413307 NZ_DS989851.1: 81897 . . . 84338 64 ZP_05027078.1 373 Microcoleus chthonoplastes PCC 7420 256377454 NC_013093.1: 3941285 . . . 3943633 65 YP_003101114.1 374 Actinosynnema mirum DSM 43827 221195188 NZ_ACFE01000002.1: 66 ZP_03568244.1 375 Atopobium rimae 241103 . . . 243577 ATCC 49626 257785020 complement(NC_013203.1: 67 YP_003180237.1 376 Atopobium 1365893 . . . 1368364) parvulum DSM 20469 227516879 complement(NZ_ACGK01000053.1: 213016 . . . 215490) 68 ZP_03946928.1 377 Atopobium vaginae DSM 15829 210630184 NZ_ABXJ01000012.1: 69 ZP_03296299.1 378 Collinsella stercoris 49466 . . . 51985 DSM 13279 41056825 AY518215.1: 989 . . . 3466 70 AAR98787.1 379 Bifidobacterium longum 223467373 NZ_ACCG01000015.1: 71 ZP_03618909.1 380 Bifidobacterium 9765 . . . 12350 breve DSM 20213 224282874 NZ_ABQP01000009.1: 72 ZP_03646196.1 381 Bifidobacterium 218668 . . . 221073 bifidum NCIMB 41171 229817819 complement(NZ_ABYS02000004.1: 73 ZP_04448101.1 382 Bifidobacterium 901411 . . . 903888) angulatum DSM 20098 212716076 complement(NZ_ABXY01000011.1: 578312 . . . 580789) 74 ZP_03324204.1 383 Bifidobacterium catenulatum DSM 16992 41056831 AY518218.1: 1430 . . . 3907 75 AAR98790.1 384 Bifidobacterium sp. CFAR 172 41056829 AY518217.1: 951 . . . 3428 76 AAR98789.1 385 Bifidobacterium pullorum 227507561 NZ_ACGF01000124.1: 77 ZP_03937610.1 386 Gardnerella 41655 . . . 44132 vaginalis ATCC 14019 261337317 NZ_ABXB03000001.1: 78 ZP_05965201.1 387 Bifidobacterium 154886 . . . 157366 gallicum DSM 20093 183601500 complement(NZ_ABOT01000001.1: 194894 . . . 197371) 79 ZP_02962870.1 388 Bifidobacterium animalis subsp. lactis HN019 41056827 AY518216.1:988 . . . 3465 80 AAR98788.1 389 Bifidobacterium pseudolongum subsp. Globosum 227516469 complement(NZ_ACGK01000047.1: 28634 . . . 31102) 81 ZP_03946518.1 390 Atopobium vaginae DSM 15829 76556241 AJ509177.1: 1 . . . 2625 82 YP_001511171.1 391 Frankia sp. EAN1pec 158318663 NC_009921.1: 8441355 . . . 8443790 83 YP_713678.1 392 Frankia alni ACN14a 111222884 complement(NC_008278.1: 84 YP_002778395.1 393 Rhodococcus 3758441 . . . 3760909) opacus B4 226360617 complement(NC_012522.1: 85 YP_701466.1 394 Rhodococcus jostii 1273076 . . . 1275478) RHA1 111018494 complement(NC_008268.1: 86 ZP_04383880.1 395 Rhodococcus 1575800 . . . 1578352) erythropolis SK121 229490027 NZ_ACNO01000014.1: 87 YP_947598.1 396 Arthrobacter 107516 . . . 109885 aurescens TC1 119962524 NC_008711.1: 2018415 . . . 2020796 88 CAD48946.1 397 Propionibacterium freudenreichii subsp. Shermanii 28868876 NC_004578.1: 1837381 . . . 1839888 89 NP_791495.1 398 Pseudomonas syringae pv. tomato str. DC3000 256425339 NC_013132.1: 8027760 . . . 8030123 90 YP_003125992.1 399 Chitinophaga pinensis DSM 2588 161075783 EU223897.1: 1 . . . 2430 91 ABX56639.1 400 Verrucomicrobiae bacterium V4 218246512 complement(NC_011726.1: 92 YP_002371883.1 401 Cyanothece sp. PCC 1758431 . . . 1760839) 8801 172055269 NC_010547.1: 390265 . . . 392673 93 YP_001806596.1 402 Cyanothece sp. ATCC 51142 126659520 complement(NZ_AAXW01000034.1: 5415 . . . 7823) 94 ZP_01730652.1 403 Cyanothece sp. CCY0110 258380665 complement(AM990467.1: 95 CAQ48286.1 404 Planktothrix 2704 . . . 5112) rubescens NIVA- CYA 98 209527806 NZ_ABYK01000067.1: 96 ZP_03276298.1 405 Arthrospira maxima 8063 . . . 10480 CS-328 196258744 NZ_ABVE01000007.1: 97 ZP_03157277.1 406 Cyanothece sp. PCC 72906 . . . 75314 7822 218440702 complement(NC_011729.1: 98 YP_002379031.1 407 Cyanothece sp. PCC 4207741 . . . 4210149) 7424 166366228 complement(NC_010296.1: 99 YP_001658501.1 408 Microcystis 3156762 . . . 3159182) aeruginosa NIES- 843 119488765 NZ_AAVU01000020.1: 100 ZP_01621774.1 409 Lyngbya sp. PCC 110903 . . . 113317 8106 17228976 complement(NC_003272.1: 101 NP_485524.1 410 Nostoc sp. PCC 1746056 . . . 1748482) 7120 254422632 NZ_DS989904.1: 4613864 . . . 4616290 102 ZP_05036350.1 411 Synechococcus sp. PCC 7335 158333641 NC_009925.1: 422232 . . . 424652 103 YP_001514813.1 412 Acaryochloris marina MBIC11017 254425820 complement(NZ_DS989905.1: 104 ZP_05039537.1 413 Synechococcus sp. 71540 . . . 74017) PCC 7335 170695087 complement(NZ_ABLD01000020.1: 33972 . . . 36368) 105 ZP_02886235.1 414 Burkholderia graminis C4D1M 209515639 complement(NZ_ABYL01000006.1: 33232 . . . 35628) 106 ZP_03264503.1 415 Burkholderia sp. H160 87303015 NZ_AANO01000008.1: 107 ZP_01085819.1 416 Synechococcus sp. 122233 . . . 124656 WH 5701 254431900 complement(NZ_DS990556.1: 108 ZP_05045603.1 417 Cyanobium sp. PCC 2146872 . . . 2149313) 7001 88808134 NZ_AAOK01000002.1: 109 ZP_01123645.1 418 Synechococcus sp. 342081 . . . 344516 WH 7805 148238545 complement(NC_009481.1: 110 YP_001223932.1 419 Synechococcus sp. 226771 . . . 229206) WH 7803 87123187 NZ_AANP01000001.1: 111 ZP_01079038.1 420 Synechococcus sp. 180603 . . . 183032 RS9917 187919971 complement(NC_010676.1: 112 YP_001889002.1 421 Burkholderia 1450148 . . . 1452541) phytofirmans PsJN 91778759 complement(NC_007952.1: 113 YP_553967.1 422 Burkholderia 1882080 . . . 1884473) xenovorans LB400 170690542 NZ_ABLD01000001.1: 114 ZP_02881709.1 423 Burkholderia 565487 . . . 567880 graminis C4D1M 209521856 NZ_ABYL01000194.1: 115 ZP_03270532.1 424 Burkholderia sp. 6778 . . . 9171 H160 186474278 complement(NC_010623.1: 116 YP_001861620.1 425 Burkholderia 2647064 . . . 2649448) phymatum STM815 225873826 complement(NC_012483.1: 117 YP_002755285.1 426 Acidobacterium 2598033 . . . 2600420) capsulatum ATCC 51196 206602403 DS995260.1: 236338 . . . 238704 118 EDZ38884.1 427 Leptospirillum sp. Group II ‘5-way CG’ 251772639 complement(GG693868.1: 119 EES53204.1 428 Leptospirillum 86578 . . . 88956) ferrodiazotrophum 56752022 complement(NC_006576.1: 120 YP_172723.1 429 Synechococcus 2156604 . . . 2158994) elongatus PCC 6301 22298729 complement(NC_004113.1: 121 NP_681976.1 430 Thermosynechococcus 1224195 . . . 1226633) elongatus BP-1 53804073 NC_002977.6: 1693459 . . . 1695894 122 YP_114037.1 431 Methylococcus capsulatus str. Bath 220907266 NC_011884.1: 1725657 . . . 1728098 123 YP_002482577.1 432 Cyanothece sp. PCC 7425 16332268 NC_000911.1: 3500713 . . . 3503178 124 NP_442996.1 433 Synechocystis sp. PCC 6803 220907424 complement(NC_011884.1: 125 YP_002482735.1 434 Cyanothece sp. PCC 1898702 . . . 1901167) 7425 241777601 complement(NZ_ACQQ01000020.1: 30393 . . . 32762) 126 ZP_04774866.1 435 Allochromatium vinosum DSM 180 114778289 NZ_AATS01000014.1: 127 ZP_01453148.1 436 Mariprofundus 23435 . . . 25801 ferrooxydans PV-1 251827471 complement(NZ_ACSD01000006.1: 128 ZP_04830548.1 437 Gallionella 39617 . . . 41986) ferruginea ES-2 121712503 XM_001273862.1: 129 XP_001273863.1 438 Aspergillus clavatus 1 . . . 2436 NRRL 1 119473535 XM_001258642.1: 130 XP_001258643.1 439 Neosartorya fischeri 1 . . . 2439 NRRL 181 169763560 XM_001727628.1: 131 XP_001727680.1 440 Aspergillus oryzae 1 . . . 2433 RIB40 145248115 XM_001396269.1: 132 XP_001396306.1 441 Aspergillus niger 1 . . . 2448 115400974 XM_001216075.1: 133 XP_001216075.1 442 Aspergillus terreus 1 . . . 2457 NIH2624 255952755 XM_002567084.1: 134 XP_002567130.1 443 Penicillium 1 . . . 2433 chrysogenum Wisconsin 54-1255 212527388 XM_002143815.1: 135 XP_002143851.1 444 Penicillium 98 . . . 2551 marneffei ATCC 18224 242783584 XM_002480171.1: 136 XP_002480216.1 445 Talaromyces 1 . . . 2448 stipitatus ATCC 10500 154321267 XM_001559899.1: 137 XP_001559949.1 446 Botryotinia 1 . . . 2466 fuckeliana B05.10 156054348 XM_001593050.1: 138 XP_001593100.1 447 Sclerotinia 1 . . . 2499 sclerotiorum 1980 189191706 XM_001932157.1: 139 XP_001932192.1 448 Pyrenophora tritici- 1 . . . 2469 repentis Pt-1C-BFP 169600613 XM_001793677.1: 140 XP_001793729.1 449 Phaeosphaeria 1 . . . 2466 nodorum SN15 58260732 XM_567776.1: 41 . . . 2545 141 XP_567776.1 450 Cryptococcus neoformans var. neoformans JEC21 46123901 XM_386504.1: 1 . . . 2460 142 XP_386504.1 451 Gibberella zeae PH-1 256732917 complement(join(GG698898.1: 143 EEU46265.1 452 Nectria 321524 . . . 322233, haematococca mpVI GG698898.1: 322285 . . . 322489, 77-13-4 GG698898.1: 322540 . . . 324081)) 225729111 FJ790496.1: 215 . . . 2677 144 ACO24516.1 453 Metarhizium anisopliae 85094948 XM_954892.2: 155 . . . 2638 145 XP_959985.1 454 Neurospora crassa OR74A 171679479 XM_001904651.1: 146 XP_001904686.1 455 Podospora anserine 1 . . . 2517 198283820 NC_011206.1: 1682860 . . . 1685307 147 YP_002220141.1 456 Acidithiobacillus ferrooxidans ATCC 53993 148243889 NC_009468.1: 90683 . . . 93145 148 YP_001220128.1 457 Acidiphilium cryptum JF-5 157364435 NC_009828.1: 1658895 . . . 1661258 149 YP_001471202.1 458 Thermotoga lettingae TMO 217966781 NC_011661.1: 369050 . . . 371428 150 YP_002352287.1 459 Dictyoglomus turgidum DSM 6724 92109503 complement(NC_007960.1: 151 YP_571790.1 460 Nitrobacter 14429 . . . 16810) hamburgensis X14 87310270 complement(NZ_AANZ01000017.1: 80191 . . . 82560) 152 ZP_01092401.1 461 Blastopirellula marina DSM 3645 152995974 NC_009654.1: 2214232 . . . 2216625 153 YP_001340809.1 462 Marinomonas sp. MWYL1 32473390 NC_005027.1: 2520925 . . . 2523306 154 NP_866384.1 463 Rhodopirellula baltica SH 1 254495580 complement(NZ_ACUL01000002.1: 21176 . . . 23557) 155 ZP_05108502.1 464 Legionella drancourtii LLAP12 254380451 NZ_DS570384.1: 88623 . . . 90992 156 ZP_04995817.1 465 Streptomyces sp. Mg1 227974767 NZ_ACGW01000133.1: 157 ZP_04023055.1 466 Lactobacillus reuteri 1172 . . . 3235 SD2112 227530011 NZ_ACGV01000134.1: 158 ZP_03960060.1 467 Lactobacillus 2320 . . . 4794 vaginalis ATCC 49540 194467185 complement(NZ_AAPZ02000001.1: 159 ZP_03073172.1 468 Lactobacillus reuteri 905298 . . . 907709) 100-23 256847586 NZ_GG698803.1: 21616 . . . 24015 160 ZP_05553031.1 469 Lactobacillus coleohominis 101-4- CHN 260662452 complement(NZ_GG704700.1: 161 ZP_05863347.1 470 Lactobacillus 145244 . . . 147643) fermentum 28-3- CHN 227903484 NZ_ACHN01000046.1: 162 ZP_04021289.1 471 Lactobacillus 59035 . . . 61452 acidophilus ATCC 4796 227877116 NZ_ACKR01000020.1: 163 ZP_03995194.1 472 Lactobacillus 11753 . . . 14191 crispatus JV-V01 227893117 NZ_ACGU01000035.1: 164 ZP_04010922.1 473 Lactobacillus 36787 . . . 39186 ultunensis DSM 16047 256844475 NZ_GG698762.1: 280846 . . . 283242 165 ZP_05549961.1 474 Lactobacillus crispatus 125-2- CHN 227521312 complement(NZ_ACGO01000008.1: 37191 . . . 39647) 166 ZP_03951361.1 475 Lactobacillus gasseri JV-V03 259501613 complement(NZ_ACLN01000019.1: 10173 . . . 12569) 167 ZP_05744515.1 476 Lactobacillus iners DSM 13335 104773655 NC_008054.1: 449229 . . . 451631 168 YP_618635.1 477 Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 227525868 NZ_ACGQ01000037.1: 169 ZP_03955917.1 478 Lactobacillus 36941 . . . 39310 jensenii JV-V16 227512366 NZ_ACGH01000107.1: 170 ZP_03942415.1 479 Lactobacillus 31655 . . . 34045 buchneri ATCC 11577 118587374 complement(NZ_AAUV01000059.1: 59008 . . . 61416) 171 ZP_01544800.1 480 Oenococcus oeni ATCC BAA-1163 28379168 complement(NC_004567.1: 172 NP_786060.1 481 Lactobacillus 2362936 . . . 2365302) plantarum WCFS1 21363093 AJ309011.1: 181 . . . 2547 173 Q937F6 482 XPKA_LACPE 81427904 NC_007576.1: 286496 . . . 288859 174 YP_394903.1 483 Lactobacillus sakei subsp. sakei 23K 116492156 NC_008525.1: 398927 . . . 401290 175 YP_803891.1 484 Pediococcus pentosaceus ATCC 25745 259648565 AP011548.1: 211570 . . . 213957 176 BAI40727.1 485 Lactobacillus rhamnosus GG 227510093 complement(NZ_ACGG01000115.1: 64541 . . . 66952) 177 ZP_03940142.1 486 Lactobacillus brevis subsp. gravesensis ATCC 27305 227891468 complement(NZ_ACGT01000007.1: 44265 . . . 46625) 178 ZP_04009273.1 487 Lactobacillus salivarius ATCC 11741 227528594 NZ_ACGS01000122.1: 179 ZP_03958643.1 488 Lactobacillus 352 . . . 2721 ruminis ATCC 25644 229542373 complement(NZ_AAWV02000001.1: 1384102 . . . 1386486) 180 ZP_04431433.1 489 Bacillus coagulans 36D1 238021480 complement(NZ_ACJW02000002.1: 913355 . . . 915730) 181 ZP_04601906.1 490 Kingella oralis ATCC 51147 259046526 NZ_ACKZ01000008.1: 182 ZP_05736927.1 491 Granulicatella 36586 . . . 38955 adiacens ATCC 49175 157150221 NC_009785.1: 333239 . . . 335623 183 YP_001449631.1 492 Streptococcus gordonii str. Challis substr. CH1 25011879 complement(NC_004368.1: 184 NP_736274.1 493 Streptococcus 1900754 . . . 1903132) agalactiae NEM316 229555065 complement(NZ_ACCR01000006.1: 74043 . . . 76418) 185 ZP_04442854.1 494 Listeria grayi DSM 20601 257866707 NZ_GG670386.1: 478278 . . . 480644 186 ZP_05646360.1 495 Enterococcus casseliflavus EC30 257870669 NZ_GG670289.1: 233512 . . . 235875 187 ZP_05650322.1 496 Enterococcus gallinarum EG2 257895654 NZ_GG670306.1: 612981 . . . 615353 188 ZP_05675307.1 497 Enterococcus faecium Com12 238810139 AP009608.1: 744956 . . . 747334 189 BAH69929.1 498 Mycoplasma fermentans PG18 193216764 NC_011025.1: 384420 . . . 386801 190 YP_002000006.1 499 Mycoplasma arthritidis 158L3-1 148377390 NC_009497.1: 136795 . . . 139182 191 YP_001256266.1 500 Mycoplasma agalactiae PG2 191639669 NC_010999.1: 2885324 . . . 2887711 192 YP_001988835.1 501 Lactobacillus casei BL23 28379861 NC_004567.1: 3169067 . . . 3171478 193 NP_786753.1 502 Lactobacillus plantarum WCFS1 227892171 complement(NZ_ACGT01000037.1: 21330 . . . 23759) 194 ZP_04009976.1 503 Lactobacillus salivarius ATCC 11741 116618551 NC_008531.1: 1449343 . . . 1451709 195 YP_818922.1 504 Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 116333142 NC_008497.1: 507704 . . . 510163 196 YP_794669.1 505 Lactobacillus brevis ATCC 367 241895257 complement(NZ_ACKU01000007.1: 101374 . . . 103833) 197 ZP_04782553.1 506 Weissella paramesenteroides ATCC 33313 170016535 NC_010471.1: 181964 . . . 184417 198 YP_001727454.1 507 Leuconostoc citreum KM20 116619034 complement(NC_008531.1: 199 YP_819405.1 508 Leuconostoc 1934181 . . . 1936622) mesenteroides subsp. mesenteroides ATCC 8293 161702316 EU255918.1: 18411 . . . 20879 200 ABX75772.1 509 Lactococcus lactis subsp. Lactis 116491770 complement(NC_008528.1: 201 YP_811314.1 510 Oenococcus oeni 1731509 . . . 1733962) PSU-1 182419955 complement(NZ_ABDT01000107.2: 13616 . . . 15991) 202 ZP_02951191.1 511 Clostridium butyricum 5521 255523324 complement(NZ_ACVI01000003.1: 203 ZP_05390294.1 512 Clostridium 55354 . . . 57747) carboxidivorans P7 15894622 NC_003030.1: 1482782 . . . 1485172 204 NP_347971.1 513 Clostridium acetobutylicum ATCC 824 226324778 complement(NZ_ABVR01000041.1: 500857 . . . 503232) 205 ZP_03800296.1 514 Coprococcus comes ATCC 27758 253580358 NZ_GG696051.1: 158015 . . . 160390 206 ZP_04857624.1 515 Ruminococcus sp. 5_1_39B_FAA 257413435 NZ_ABYJ02000055.1: 207 ZP_04743029.2 516 Roseburia 10320 . . . 12779 intestinalis L1-82 154500233 complement(NZ_AAXG02000041.1: 34174 . . . 36609) 208 ZP_02038271.1 517 Bacteroides capillosus ATCC 29799 219119570 XM_002180506.1: 209 XP_002180542.1 518 Phaeodactylum 1 . . . 2508 tricornutum CCAP 1055/1 91975971 NC_007958.1: 1660408 . . . 1662762 210 YP_568630.1 519 Rhodopseudomonas palustris BisB5 86750966 complement(NC_007778.1: 211 YP_487462.1 520 Rhodopseudomonas 4411322 . . . 4413676) palustris HaA2 39934743 NC_005296.1: 1858439 . . . 1860790 212 NP_947019.1 521 Rhodopseudomonas palustris CGA009 90425290 complement(NC_007925.1: 213 YP_533660.1 522 Rhodopseudomonas 4235875 . . . 4238229) palustris BisB18 121583071 NC_008758.1: 65532 . . . 67904 214 YP_973512.1 523 Polaromonas naphthalenivorans CJ2 115376972 complement(NZ_AAMD01000095.1: 4173 . . . 6533) 215 ZP_01464191.1 524 Stigmatella aurantiaca DW4/3-1 148547676 complement(NC_009512.1: 216 YP_001267778.1 525 Pseudomonas putida 2807645 . . . 2810020) F1 116668711 NC_008541.1: 145493 . . . 147928 217 YP_829644.1 526 Arthrobacter sp. FB24 220911083 NC_011886.1: 321712 . . . 324174 218 YP_002486392.1 527 Arthrobacter chlorophenolicus A6 260517200 complement(NZ_ABUN01000002.1: 92936 . . . 95461) 219 ZP_05816651.1 528 Sanguibacter keddieii DSM 10542 229821527 NC_012669.1: 3398743 . . . 3401217 220 YP_002883053.1 529 Beutenbergia cavernae DSM 12333 256832813 NC_013174.1: 1712156 . . . 1714588 221 YP_003161540.1 530 Jonesia denitrificans DSM 20603 227428425 complement(NZ_ABVC01000008.1: 152502 . . . 154988) 222 ZP_03911482.1 531 Xylanimonas cellulosilytica DSM 15894 165929357 AM182260.1: 1 . . . 2481 223 CAJ57850.1 532 Cellulomonas flavigena 145223927 NC_009338.1: 3525804 . . . 3528275 224 YP_001134605.1 533 Mycobacterium gilvum PYR-GCK 120404048 NC_008726.1: 3236585 . . . 3239083 225 YP_953877.1 534 Mycobacterium vanbaalenii PYR-1 257069356 NC_013172.1: 2493744 . . . 2496215 226 YP_003155611.1 535 Brachybacterium faecium DSM 4810 256824167 NC_013169.1: 273585 . . . 276047 227 YP_003148127.1 536 Kytococcus sedentarius DSM 20547 148271607 NC_009480.1: 506602 . . . 509040 228 YP_001221168.1 537 Clavibacter michiganensis subsp. michiganensis NCPPB 382 145594129 complement(NC_009380.1: 229 YP_001158426.1 538 Salinispora tropica 1798516 . . . 1800918) CNB-440 159037167 complement(NC_009953.1: 230 YP_001536420.1 539 Salinispora 1767167 . . . 1769569) arenicola CNS-205 238063593 complement(NZ_GG657738.1: 231 ZP_04608302.1 540 Micromonospora sp. 5405062 . . . 5407251) ATCC 39149 118469963 NC_008596.1: 3674267 . . . 3676639 232 YP_887914.1 541 Mycobacterium smegmatis str. MC2 155 108799759 NC_008146.1: 2939527 . . . 2941947 233 YP_639956.1 542 Mycobacterium sp. MCS 240170498 complement(NZ_ACBV01000039.1: 21 . . . 2423) 234 ZP_04749157.1 543 Mycobacterium kansasii ATCC 12478 183982748 complement(NC_010612.1: 235 YP_001851039.1 544 Mycobacterium 3341817 . . . 3344219) marinum M 41407671 complement(NC_002944.2: 236 NP_960507.1 545 Mycobacterium 1726717 . . . 1729131) avium subsp. paratuberculosis K- 10 254819329 NZ_ABIN01000047.1: 237 ZP_05224330.1 546 Mycobacterium 36474 . . . 38837 intracellulare ATCC 13950 169629591 complement(NC_010397.1: 238 YP_001703240.1 547 Mycobacterium 2559451 . . . 2561871) abscessus 84496279 complement(NZ_AAMN01000002.1: 433314 . . . 435707) 239 ZP_00995133.1 548 Janibacter sp. HTCC2649 72163369 NC_007333.1: 3478272 . . . 3480650 240 YP_291026.1 549 Thermobifida fusca YX 227984600 complement(NZ_ABUZ01000013.1: 70531 . . . 72909) 241 ZP_04031845.1 550 Thermomonospora curvata DSM 43183 229855558 complement(NZ_ABUU01000106.1: 1385 . . . 3700) 242 ZP_04475514.1 551 Streptosporangium roseum DSM 43021 229209207 NZ_ABUI01000028.1: 243 ZP_04335641.1 552 Nocardiopsis 55841 . . . 58189 dassonvillei subsp. dassonvillei DSM 43111 229862587 NZ_ABUS01000001.1: 244 ZP_04482201.1 553 Stackebrandtia 1911934 . . . 1914330 nassauensis DSM 44728 256376052 NC_013093.1: 2125566 . . . 2127935 245 YP_003099712.1 554 Actinosynnema mirum DSM 43827 32141117 complement(NC_003888.3: 246 NP_733508.1 555 Streptomyces 656319 . . . 658772) coelicolor A3(2) 117164830 complement(AM238664.1: 247 CAJ88379.1 556 Streptomyces 846551 . . . 849055) ambofaciens ATCC 23877 256811868 complement(NZ_ACFA01000015.1: 248 ZP_05536883.1 557 Streptomyces 3377 . . . 5761) griseoflavus Tu4000 254405496 complement(NZ_DS570938.1: 249 ZP_05020421.1 558 Streptomyces 43288 . . . 45726) sviceus ATCC 29083 260644540 complement(FN554889.1: 250 CBG67625.1 559 Streptomyces 480316 . . . 482694) scabiei 87.22 29827814 complement(NC_003155.4: 251 NP_822448.1 560 Streptomyces 1579336 . . . 1581717) avermitilis MA- 4680 239932594 NZ_ABYA01000503.1: 252 ZP_04689547.1 561 Streptomyces 5217 . . . 7595 ghanaensis ATCC 14672 256800397 complement(NZ_ACEZ01000048.1: 253 ZP_05530021.1 562 Streptomyces 24916 . . . 27303) viridochromogenes DSM 40736 256774038 complement(NZ_ACEX01000074.1: 46221 . . . 48614) 254 ZP_05512501.1 563 Streptomyces hygroscopicus ATCC 53653 260452518 NZ_ACZH01000001.1: 255 ZP_05800927.1 564 Streptomyces 321972 . . . 324359 flavogriseus ATCC 33331 182440556 NC_010572.1: 8084439 . . . 8086826 256 YP_001828275.1 565 Streptomyces griseus subsp. griseus NBRC 13350 239982969 NZ_ABYC01000425.1: 257 ZP_04705493.1 566 Streptomyces albus 13265 . . . 15646 J1074 254381599 NZ_DS570386.1: 118817 . . . 121204 258 ZP_04996963.1 567 Streptomyces sp. Mg1 256674998 NZ_ACEU01000020.1: 259 ZP_05485309.1 568 Streptomyces sp. 1507 . . . 3900 SPB78 227377421 NZ_ABUC01000002.1: 260 ZP_03860882.1 569 Kribbella flavida 229225 . . . 231603 DSM 17836 54023297 complement(NC_006361.1: 261 YP_117539.1 570 Nocardia farcinica 1487629 . . . 1490097) IFM 10152 158313048 NC_009921.1: 1426213 . . . 1428621 262 YP_001505556.1 571 Frankia sp. EAN1pec 86742227 complement(NC_007777.1: 263 YP_482627.1 572 Frankia sp. CcI3 4238578 . . . 4240986) 256395329 NC_013131.1: 7133131 . . . 7135533 264 YP_003116893.1 573 Catenulispora acidiphila DSM 44928 117927729 NC_008578.1: 555555 . . . 557948 265 YP_872280.1 574 Acidothermus cellulolyticus 11B 119717842 complement(NC_008699.1: 266 YP_924807.1 575 Nocardioides sp. 3839565 . . . 3841961) JS614 134098496 NC_009142.1: 2098116 . . . 2100512 267 YP_001104157.1 576 Saccharopolyspora erythraea NRRL 2338 209550756 NC_011369.1: 3264963 . . . 3267347 268 YP_002282673.1 577 Rhizobium leguminosarum bv. trifolii WSM2304 241206160 NC_012850.1: 3503904 . . . 3506288 269 YP_002977256.1 578 Rhizobium leguminosarum bv. trifolii WSM1325 190893254 NC_010994.1: 3714233 . . . 3716620 270 YP_001979796.1 579 Rhizobium etli CIAT 652 86359034 NC_007761.1: 3623921 . . . 3626308 271 YP_470926.1 580 Rhizobium etli CFN 42 222081270 complement(NC_011983.1: 272 YP_002540633.1 581 Agrobacterium 490969 . . . 493383) radiobacter K84 254720555 NZ_ACBQ01000064.1: 273 ZP_05182366.1 582 Brucella sp. 83/13 129340 . . . 131718 239835057 complement(NZ_ACQA01000003.1: 10528 . . . 13017) 274 ZP_04683384.1 583 Ochrobactrum intermedium LMG 3301 153012043 NC_009671.1: 16319 . . . 18706 275 YP_001373254.1 584 Ochrobactrum anthropi ATCC 49188 146339061 complement(NC_009445.1: 276 YP_001204109.1 585 Bradyrhizobium sp. 2141749 . . . 2144226) ORS278 148253833 complement(NC_009485.1: 277 YP_001238418.1 586 Bradyrhizobium sp. 2424642 . . . 2427059) BTAi1 27377629 complement(NC_004463.1: 278 NP_769158.1 587 Bradyrhizobium 2749734 . . . 2752139) japonicum USDA 110 92117435 complement(NC_007964.1: 279 YP_577164.1 588 Nitrobacter 2109162 . . . 2111570) hamburgensis X14 240137143 NC_012808.1: 407982 . . . 410417 280 YP_002961612.1 589 Methylobacterium extorquens AM1 110634584 complement(NC_008254.1: 281 YP_674792.1 590 Mesorhizobium sp. 2388345 . . . 2390747) BNC1 260467447 NZ_ACZA01000051.1: 282 ZP_05813617.1 591 Mesorhizobium 15952 . . . 18360 opportunistum WSM2075 75676138 NC_007406.1: 2135469 . . . 2137856 283 YP_318559.1 592 Nitrobacter winogradskyi Nb- 255 170749020 complement(NC_010505.1: 284 YP_001755280.1 593 Methylobacterium 2769888 . . . 2772470) radiotolerans JCM 2831 170746859 complement(NC_010505.1: 285 YP_001753119.1 594 Methylobacterium 465997 . . . 468552) radiotolerans JCM 2831 254558916 NC_012988.1: 271224 . . . 273809 286 YP_003066011.1 595 Methylobacterium extorquens DM4 240140298 NC_012808.1: 3931130 . . . 3933676 287 YP_002964777.1 596 Methylobacterium extorquens AM1 220925990 NC_011894.1: 6291823 . . . 6294321 288 YP_002501292.1 597 Methylobacterium nodulans ORS 2060 220919962 complement(NC_011892.1: 289 YP_002495265.1 598 Methylobacterium 451339 . . . 453840) nodulans ORS 2060 170741732 NC_010511.1: 3988668 . . . 3991166 290 YP_001770387.1 599 Methylobacterium sp. 4-46 239815802 complement(NC_012791.1: 291 YP_002944712.1 600 Variovorax 2971257 . . . 2973608) paradoxus S110 89069402 NZ_AAOT01000017.1: 292 ZP_01156757.1 601 Oceanicola 31124 . . . 33460 granulosus HTCC2516 119509641 complement(NZ_AAVW01000007.1: 15878 . . . 18259) 293 ZP_01628787.1 602 Nodularia spumigena CCY9414 186682350 NC_010628.1: 2389837 . . . 2392218 294 YP_001865546.1 603 Nostoc punctiforme PCC 73102 75906719 complement(NC_007413.1: 295 YP_321015.1 604 Anabaena variabilis 617971 . . . 620352) ATCC 29413 225522346 NZ_ACIR01000182.1: 296 ZP_03769140.1 605 Nostoc azollae' 0708 624 . . . 2756 37520566 NC_005125.1: 1065716 . . . 1068097 297 NP_923943.1 606 Gloeobacter violaceus PCC 7421 86608623 NC_007776.1: 1182311 . . . 1184686 298 YP_477385.1 607 Synechococcus sp. JA-2-3B′a(2-13) 150398192 complement(NC_009636.1: 299 YP_001328659.1 608 Sinorhizobium 3144485 . . . 3146857) medicae WSM419 116249832 complement(NC_008380.1: 300 YP_765670.1 609 Rhizobium 82152 . . . 84617) leguminosarum bv. viciae 3841 195970218 complement(NC_003047.1: 301 NP_384212.2 610 Sinorhizobium 123688 . . . 126141) meliloti 1021 171912985 NZ_ABIZ01000001.1: 302 ZP_02928455.1 611 Verrucomicrobium 4370841 . . . 4373354 spinosum DSM 4136 163849496 complement(NC_010172.1: 303 YP_001637539.1 612 Methylobacterium 46285 . . . 48720) extorquens PA1 85714839 NZ_AAMY01000005.1: 304 ZP_01045825.1 613 Nitrobacter sp. Nb- 72844 . . . 75210 311A 168704325 complement(NZ_ABGO01000323.1: 57 . . . 2462) 305 ZP_02736602.1 614 Gemmata obscuriglobus UQM 2246 256829143 complement(NC_013173.1: 306 YP_003157871.1 615 Desulfomicrobium 1530488 . . . 1532881) baculatum DSM 4028 223939426 complement(NZ_ABOX02000044.1: 41392 . . . 43863) 307 ZP_03631304.1 616 bacterium Ellin514 237747078 complement(NZ_GG658151.1: 308 ZP_04577558.1 617 Oxalobacter 2005797 . . . 2008190) formigenes HOxBLS 237749232 complement(NZ_GG658170.1: 309 ZP_04579712.1 618 Oxalobacter 2042015 . . . 2044411) formigenes OXCC13 116624013 NC_008536.1: 6218168 . . . 6220537 310 YP_826169.1 619 Solibacter usitatus Ellin6076 194336959 complement(NC_011060.1: 311 YP_002018753.1 620 Pelodictyon 2004498 . . . 2006885) phaeoclathratiforme BU-1 194334425 complement(NC_011059.1: 312 YP_002016285.1 621 Prosthecochloris 1762093 . . . 1764489) aestuarii DSM 271 189346840 complement(NC_010803.1: 313 YP_001943369.1 622 Chlorobium 1427679 . . . 1430054) limicola DSM 245 21674344 complement(NC_002932.3: 314 NP_662409.1 623 Chlorobium 1423776 . . . 1426289) tepidum TLS 110597897 complement(NZ_AASE01000009.1: 315 ZP_01386179.1 624 Chlorobium 37756 . . . 40179) ferrooxidans DSM 13031 78187379 complement(NC_007512.1: 316 YP_375422.1 625 Chlorobium 1709621 . . . 1712050) luteolum DSM 273 71907690 complement(NC_007298.1: 317 YP_285277.1 626 Dechloromonas 2220090 . . . 2222456) aromatica RCB 74316849 NC_007404.1: 876540 . . . 878978 318 YP_314589.1 627 Thiobacillus denitrificans ATCC 25259 91775246 complement(NC_007947.1: 319 YP_545002.1 628 Methylobacillus 935825 . . . 938200) flagellatus KT 30250069 NC_004757.1: 2318109 . . . 2320481 320 NP_842139.1 629 Nitrosomonas europaea ATCC 19718 114332052 NC_008344.1: 2209596 . . . 2211971 321 YP_748274.1 630 Nitrosomonas eutropha C91 82702122 NC_007614.1: 1152112 . . . 1154535 322 YP_411688.1 631 Nitrosospira multiformis ATCC 25196 77166175 NC_007484.1: 3082455 . . . 3084869 323 YP_344700.1 632 Nitrosococcus oceani ATCC 19707 46445639 complement(NC_005861.1: 324 YP_007004.1 633 Candidatus 5907 . . . 8303) Protochlamydia amoebophila UWE25 16263040 complement(NC_003037.1: 325 NP_435833.1 634 Sinorhizobium 591065 . . . 593440) meliloti 1021 229532493 NZ_ABUV01000006.1: 326 ZP_04421874.1 635 Sulfurospirillum 47234 . . . 49585 deleyianum DSM 6946 13475490 NC_002678.2: 5384229 . . . 5386652 327 NP_107054.1 636 Mesorhizobium loti MAFF303099 209885940 complement(NC_011386.1: 328 YP_002289797.1 637 Oligotropha 2786353 . . . 2788746) carboxidovorans OM5 182679166 NC_010581.1: 2524033 . . . 2526420 329 YP_001833312.1 638 Beijerinckia indica subsp. indica ATCC 9039

Numerous examples of polynucleotides, genes and/or polypeptides encoding phosphotransacetylase are known in the art and can be used in relation to the recombinant host cells disclosed herein. In embodiments, the phosphotransacetylase can be EutD from Lactobacillus plantarum. In embodiments, the phosphotransacetylase can be the phosphotransacetylase from Bacillus subtilis. This phosphotransacetylase has a specific activity of 1371 μmol/min/mg and a Km 0.06 mM for acetyl-CoA (Rado and Hoch, Biochim. Biophys. Acta. 321: 114-25; 1973). In addition, the equilibrium constant (Keq) of this reaction was found to be 154±14 in favor of the formation of acetyl-CoA according to the following formula:

$\frac{\left\lbrack {{acetyl}\text{-}{CoA}} \right\rbrack\lbrack{Pi}\rbrack}{\lbrack{CoA}\rbrack\left\lbrack {{acetyl}\text{-}P} \right\rbrack} = {Keq}$

In embodiments, host cells comprise a polypeptide having at least about 80%, at least about 85%, at least about 90%, or at least about 99% identity to a polypeptide of Table 10 or an active fragment thereof or a polynucleotide encoding such a polypeptide. In embodiments, the phosphotransacetylase can be a polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identity to SEQ ID NO: 1472 or an active fragment thereof. In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphotransacetylase can include, but is not limited to, a sequence provided in the following tables 10 or 12.

TABLE 10 SEQ ID NOs of phosphotransacetylase target gene coding regions and proteins. SEQ ID SEQ ID NO: NO: Nucleic Amino Description acid acid Amino acid sequence EutD 1111 1472 MDLFESLAQKITGKDQTIVFPEGTEPRIVGAAARLAADGLVKPIVLGATDKVQAVANDLN phosphotransacetylase ADLTGVQVLDPATYPAEDKQAMLDALVERRKGKNTPEQAAKMLEDENYFGTMLVYMGKAD from Lactobacillus GMVSGAIHPTGDTVRPALQIIKTKPGSHRISGAFIMQKGEERYVFADCAINIDPDADTLA plantarum EIATQSAATAKVFDIDPKVAMLSFSTKGSAKGEMVTKVQEATAKAQAAEPELAIDGELQF DAAFVEKVGLQKAPGSKVAGHANVFVFPELQSGNIGYKIAQRFGHFEAVGPVLQGLNKPV SDLSRGCSEEDVYKVAIITAAQGLA Phosphotransacetylase 1061 1422 MADLFSTVQEKVAGKDVKIVFPEGLDERILEAVSKLAGNKVLNPIVIGNENEIQAKAKEL from Bacillus subtilis NLTLGGVKIYDPHTYEGMEDLVQAFVERRKGKATEEQARKALLDENYFGTMLVYKGLADG LVSGAAHSTADTVRPALQIIKTKEGVKKTSGVFIMARGEEQYVFADCAINIAPDSQDLAE IAIESANTAKMFDIEPRVAMLSFSTKGSAKSDETEKVADAVKIAKEKAPELTLDGEFQFD AAFVPSVAEKKAPDSEIKGDANVFVFPSLEAGNIGYKIAQRLGNFEAVGPILQGLNMPVN DISRGCNAEDVYNLALITAAQAL

Additional suitable phosphotransacetylase target gene coding regions and proteins were identified by diversity searching and clustering. A blast search of the non redundant GenBank protein database (NR) was performed with the L. plantarum EutD protein as a query sequence. A blast cut-off (Evalue) of 0.01 was applied. This search resulted in 2124 sequence matches. Redundancy reduction was achieved by clustering proteins with the CD-HIT program with parameters set at 95% sequence identity and 90% length overlap. The longest seed sequence, representative of each cluster, is retained for further analysis. Clustering reduced the number of protein sequences to 1336. Further clean-up of the sequences by removing sequences<280 amino acids and sequences>795 amino acids resulted in 1231 seqs.

The Brenda database was queried for experimentally verified phosphate acetyltransferases. Thirteen were found in the following organisms: S. enterica, E. coli K12, V. Parvula, C. Kluyveri, C. Acetobutylicum, C. Thermocellum, M. thermophila, S. pyogenes, B. subtilis, L. fermentum, L. plantarum, L. sanfranciscensis, B. subtilis, L. fermentum, L. plantarum, L. sanfranciscensis, R. palustris, E. coli.

Experimentally verified phosphate acetyltransferases (EC 2.3.1.8) belong to the PTA_PTB pfam family. However, the PTA_PTB domain is present in 13 distinct architectures (http://pfam.janelia.org/family/PF01515, Pfam database version 24). The motivation for investigating the domain architecture is to determine which of the proteins, that were identified by BLAST search, are likely to be phosphate acetyltransferases. Experimentally verified sequences extracted from the BRENDA database as well as sequences retained after the CD-HIT clustering and clean-up, were searched against the Pfam database to determine their domain architecture. Pfam is a collection of multiple sequence alignments and profile hidden Markov models (HMMs). Each Pfam HMM represents a protein family or domain. By searching a protein sequence against the Pfam library of HMMs, it is possible to determine which domains it carries i.e. its domain architecture. (The Pfam protein families database: R. D. Finn, J. Tate, J. Mistry, P. C. Coggill, J. S. Sammut, H. R. Hotz, G. Ceric, K. Forslund, S. R. Eddy, E. L. Sonnhammer and A. Bateman Nucleic Acids Research (2008) Database Issue 36:D281-D288)

Twelve of the experimentally verified proteins only contained the PTA_PTB domain. Two sequences, from R. palustris and E. coli, contained two domains PTA_PTB and DRTGG, a domain of unknown function. Therefore, from the CD-HIT clustering results, proteins that contained either the PTA_PTB domain only (Group 1: 549 sequences) or a combination of PTA_PTB+DRTGG domains (Group 2: 201 sequences) were chosen.

Furthermore, the PTA_PTB domain, as the name indicates, is actually not specific to phosphate acetyltransferases. The domain is also a signature for phosphate butyryltransferases (EC 2.3.1.19). Two methods to distinguish between the two subfamilies: acetyltransferases and butyryltransferases were employed and are as follows:

To further characterize the relationship among the sequences, multiple sequence alignment MSA), phylogenetic analysis, profile HMMs and GroupSim analysis were performed. For this set of analyses, the phosphate acetyltransferases are split in two groups. Group 1 contains phosphate acetyltransferases with the PTA_PTB domain only, while Group 2 contains phosphate acetyltransferases with PTA_PTB+DRTGG. The motivation here is to generate groups with similar lengths.

Clustal X, version 2.0 was used for sequence alignments with default parameters. (Thompson J D, et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. (1997) 25:4876-4882.)

Alignment results were utilized to compute % sequence identities to a reference sequence. If the sequence from L. plantarum is taken as a reference, % IDs range from as low as 10.5% to 75.6% for the closest sequence. Alignment results also provided the basis for re-constructing phylogenetic trees. The Neighbor Joining method, available in the Jalview package version 2.3, was used to produce the trees, and computed trees were visualized in MEGA 4 (Tamura, Dudley, Nei, and Kumar 2007). The Neighbor Joining method is a method for re-constructing phylogenetic trees, and computing the lengths of the branches of this tree. In each stage, the two nearest nodes of the tree (the term “nearest nodes” will be defined in the following paragraphs) are chosen and defined as neighbors in our tree. This is done recursively until all of the nodes are paired together. “The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 July; 4(4):406-25. Saitou N, Nei M.” Jalview Version 2 is a system for interactive editing, analysis and annotation of multiple sequence alignments (Waterhouse, A. M., Procter, J. B., Martin, D. M. A, Clamp, M. and Barton, G. J. (2009) “Jalview Version 2—a multiple sequence alignment editor and analysis workbench” Bioinformatics 25 (9) 1189-1191). The MEGA software provides tools for exploring, discovering, and analyzing DNA and protein sequences from an evolutionary perspective. MEGA4 enables the integration of sequence acquisition with evolutionary analysis. It contains an array of input data and multiple results explorers for visual representation; the handling and editing of sequence data, sequence alignments, inferred phylogenetic trees; and estimated evolutionary distances. The results explorers allow users to browse, edit, summarize, export, and generate publication-quality captions for their results. MEGA 4 also includes distance matrix and phylogeny explorers as well as advanced graphical modules for the visual representation of input data and output results (Tamura K, Dudley J, Nei M & Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599).

Taken together, % IDs and the generated tree (FIG. 4) indicated that potential phosphate acetyltransferases (PTA_PTB domain only) are divided in two major subfamilies. Subfamily 1 from 10.5% ID to ˜20% ID (176 sequences) and Subfamily 2 from ˜20% ID to 75.6% ID (361 sequences). The third Subfamily, of 12 sequences, has % ID ranging from 17% ID to 25% ID with respect to the L. plantarum sequence.

Based on experimentally verified sequences contained within each of the Subfamilies, Subfamily 1 and Subfamily 2 were determined to represent phosphate butyryltransferases (PTB) and phosphate acetyltransferases (PTA) respectively.

Discrimination between Subfamily 1 members and Subfamily 2 members was also performed by GroupSim analysis (Capra and Singh (2008) Bioinformatics 24: 1473-1480). The GroupSim method identifies amino acid residues that determine a protein's functional specificity. In a multiple sequence alignment (MSA) of a protein family whose sequences are divided into multiple Subfamilies, amino acid residues that distinguish between the functional Subfamilies of sequences can be identified. The method takes a multiple sequence alignment (MSA) and known specificity groupings as input, and assigns a score to each amino acid position in the MSA. Higher scores indicate a greater likelihood that an amino acid position is a specificity determining position (SDP).

GroupSim analysis performed on the MSA of 537 sequences (divided into Subfamily 1 and Subfamily 2 by the phylogenetic analysis above) identified highly discriminating positions. Listed in Table 11 are positions (Pos) having scores greater than to 0.7, where a perfect score of 1.0 would indicate that all proteins within the Subfamily have the listed amino acid in the specified position and between Subfamilies the amino acid would always be different. The “Pattern” columns give the amino acid(s) in single letter code. Numbers between parentheses indicate frequency of occurrence of each amino acid at the particular position. The amino acid position number in column 1 is for the PTA protein sequence from Lactobacillus plantarum, the representative protein of group 2 with a GI#28377658 (SEQ ID NO: 1472).

TABLE 11 Highly discriminating amino acid positions for Subfamily 1 (PTB) and Subfamily 2 (PTA) from GroupSim analysis. Pos Score Pattern PTB Pattern PTA 212 0.980314 Group 1: E(173), D(2), Group 2: S(360), N(1) L(1) 305 0.87236 Group 1: L(152), V(11), Group 2: D(360), Q(1) M(5), I(5), F(3) 242 0.831201 Group 1: A(142), D(15), Group 2: Q(361) S(13), G(4), T(2) 208 0.776954 Group 1: L(130), I(35), Group 2: S(355), A(6) V(11) 125 0.705868 Group 1: K(175), R(1) Group 2: S(215), A(85), G(41), C(14), N(4), T(2)

An alternative structure/function characterization of the PTA and PTB subfamilies of enzymes was performed using the HMMER software package (the theory behind profile HMMs is described in R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998; Krogh et al., 1994; J. Mol. Biol. 235:1501-1531), following the user guide which is available from HMMER (Janelia Farm Research Campus, Ashburn, Va.).

Using a multiple sequence alignment of the experimentally verified sequences (containing the PTA_PTB domain only) in Subfamily 2, a profile Hidden Markov Model (HMM) was created for representing Subfamily 2 members. The sequences were:

-   -   1. BAB19267.1 from Lactobacillus sanfranciscensis (SEQ ID NO:         1475)     -   2. NP_784550.1 from Lactobacillus plantarum WCFS1 (SEQ ID NO:         1472)     -   3. ZP_03944466.1 from Lactobacillus fermentum ATCC 14931 (SEQ ID         NO: 1453)     -   4. NP_391646.1 from Bacillus subtilis subsp. subtilis str. 168         (SEQ ID NO: 1422)     -   5. AAA72041.1 from Methanosarcina thermophila (SEQ ID NO: 1277)     -   6. ZP_03152606.1 from Clostridium thermocellum DSM 4150 (SEQ ID         NO: 1275)     -   7. NP_348368.1 from Clostridium acetobutylicum ATCC 824 (SEQ ID         NO: 1206)     -   8. YP_001394780.1 from Clostridium kluyveri DSM 555 (SEQ ID NO:         1200)     -   9. ZP_03855267.1 from Veillonella parvula DSM 2008 (SEQ ID NO:         1159)     -   10. YP_149725.1 from Salmonella enterica subsp. enterica serovar         Paratyphi A str. ATCC 9150 (SEQ ID NO: 1129).

The Profile HMM was built as follows: The 10 seed sequences (sequences representing experimentally verified function) that are in Subfamily 2 were aligned using Clustal X (interface to Clustal W) with default parameters. The hmmbuild program was run on each set of the aligned sequences using default parameters. hmmbuild reads the multiple sequence alignment file, builds a new Profile HMM, and saves the Profile HMM to file. Using this program an un-calibrated profile was generated from the multiple alignment for each set of subunit sequences described above.

The Profile HMM was read using hmmcalibrate which scores a large number of synthesized random sequences with the Profile (the default number of synthetic sequences used is 5,000), fits an extreme value distribution (EVD) to the histogram of those scores, and re-saves the HMM file now including the EVD parameters. These EVD parameters (μ and λ) are used to calculate the E-values of bit scores when the profile is searched against a protein sequence database. hmmcalibrate writes two parameters into the HMM file on a line labeled “EVD”: these parameters are the μ (location) and λ (scale) parameters of an extreme value distribution (EVD) that best fits a histogram of scores calculated on randomly generated sequences of about the same length and residue composition as SWISS-PROT. This calibration was done once for the Profile HMM.

The calibrated profile HMM for the Subfamily 2 set is provided as Table 14. The Profile HMM table gives the probability of each amino acid occurring at each position in the amino acid sequence. The amino acids are represented by the one letter code. The first line for each position reports the match emission scores: probability for each amino acid to be in that state (highest score is highlighted). The second line reports the insert emission scores, and the third line reports on state transition scores: M→M, M→I, M→D; I→M, I→I; D→M, D→D; B→M; M→E. Table 14 shows that in the Subfamily 2 profile HMM, methionine has a 3792 ans 4481 probability of being in the first two positions.

The Subfamily 2 profile HMM was evaluated using hmmsearch, with the Z parameter set to one billion, for the ability to discriminate Subfamily 1 members from those of Subfamily 2. The hmmsearch program takes the hmm file for the Subfamily 2 profile HMM and all the sequences from both Subfamilies and assigns an E-value score to each sequence. This E-value score is a measure of fit to the Profile HMM, with a lower score being a better fit. The Profile HMM distinguished Subfamily 2 members from Subfamily 1 members and there was a large margin of E-value difference between the worst scoring Subfamily 2 member (5e-34) and the best scoring Subfamily 1 member (4.3e-07). This analysis shows that the Profile HMM prepared for Subfamily 2 phosphate acetyltransferases (PTA) distinguishes PTA sequences from phosphate butyryltransferase PTB protein sequences.

Based on these analyses, 361 phosphate acetyltransferase sequences (PTA_PTB domain only) were identified and are provided in Table 12a.

TABLE 12a SEQ ID NOs of phosphotransacetylase target gene coding regions and proteins GENBANK Nucleic GENBANK Amino Nucleotide Acid Amino Acid Acid GI Sequence SEQ Sequence SEQ Source Number Accession Information ID NO: Accession No. ID NO: Organism 255994631 complement 762 ZP_05427766.1 1123 Eubacterium (NZ_ACON01000003.1: saphenum 407639 . . . 408607) ATCC 49989 223935781 NZ_ABOX02000007.1: 763 ZP_03627696.1 1124 bacterium 10458 . . . 11600 Ellin514 196232920 NZ_ABVL01000018.1: 764 ZP_03131770.1 1125 Chthoniobacter 134406 . . . 135449 flavus Ellin428 187735919 NC_010655.1: 765 YP_001878031.1 1126 Akkermansia 1714393 . . . 1715484 muciniphila ATCC BAA-835 237732443 complement 766 ZP_04562924.1 1127 Citrobacter (NZ_GG657366.1: sp. 30_2 2437071 . . . 2438087 157144617 NC_009792.1: 767 YP_001451936.1 1128 Citrobacter 326993 . . .328009 koseri ATCC BAA-895 56412650 NC_006511.1 768 YP_149725.1 1129 Salmonella 473496 . . . 474512 enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 161502384 NC_010067.1: 769 YP_001569496.1 1130 Salmonella 425125 . . . 426141 enterica subsp. arizonae serovar 62:z4, z23:-- 16130383 complement 770 NP_416953.1 1131 Escherichia (NC_000913.2: coli str. K-12 2570511 . . . 2571527) substr. MG1655 238895918 complement 771 YP_002920654.1 1132 Klebsiella (NC_012731.1: pneumoniae 3863205 . . . 3864221) NTUH-K2044 238794182 complement 772 ZP_04637797.1 1133 Yersinia (NZ_AALF02000025.1: intermedia 17041 . . . 18039) ATCC 29909 90414632 complement 773 ZP_01222604.1 1134 Photobacterium (NZ_AAPH01000046.1: profundum 12550 . . . 13527) 3TCK 163749608 complement 774 ZP_02156855.1 1135 Shewanella (NZ_ABIC01000008.1: benthica 73942 . . . 74913) KT99 120554157 NC_008740.1: 775 YP_958508.1 1136 Marinobacter 1389827 . . . 1390810 aquaeolei VT8 51246887 complement 776 YP_066771.1 1137 Desulfotalea (NC_006138.1: psychrophila 3433697 . . . 3434677) LSv54 226362753 complement 777 YP_002780531.1 1138 Rhodococcus (NC_012522.1: opacus 3613049 . . . 3614074) B4 111020534 complement 778 YP_703506.1 1139 Rhodococcus (NC_008268.1: jostii 3751557 . . . 3752585) RHA1 256669010 NZ_ACEV01000044.1: 779 ZP_05479963.1 1140 Streptomyces 80096 . . . 81085 sp. AA4 226227292 complement 780 YP_002761398.1 1141 Gemmatimonas (NC_012489.1: aurantiaca 2203452 . . . 2204474) T-27 239627158 complement 781 ZP_04670189.1 1142 Clostridiales (NZ_DS990260.1: bacterium 924717 . . . 925736) 1_7_47FAA 256753163 complement 782 ZP_05493958.1 1143 Clostridium (NZ_ACXX01000001.1: papyrosolvens 167634 . . . 168635) DSM 2782 257063834 NC_013165.1: 783 YP_003143506.1 1144 Slackia 1278820 . . 1279818 heliotrinireducens DSM 20476 254477436 NZ_DS999054.1: 784 ZP_05090822.1 1145 Ruegeria 3384063 . . . 3384914 sp. R11 126732220 complement 785 ZP_01748021.1 1146 Sagittula (NZ_AAYA01000016.1: stellata 68052 . . . 68996) E-37 19704507 complement 786 NP_604069.1 1147 Fusobacterium (NC_003454.1: nucleatum subsp. 1833702 . . . 1834715) nucleatum ATCC 25586 260494604 NZ_GG704456.1: 787 ZP_05814734.1 1148 Fusobacterium 222376 . . . 223389 sp. 3_1_33 262067001 complement 788 ZP_06026613.1 1149 Fusobacterium (NZ_ACJY01000064.1: periodonticum 9865 . . . 10869) ATCC 33693 257452333 complement 789 ZP_05617632.1 1150 Fusobacterium (NZ_ACDD01000037.1: sp. 3_1_5R 1250 . . . 2263) 257463639 NZ_ACDG01000104.1: 790 ZP_05628030.1 1151 Fusobacterium 17109 . . . 18122 sp. D12 253583748 complement 791 ZP_04860946.1 1152 Fusobacterium (NZ_GG696122.1: varium 645905 . . . 646912) ATCC 27725 237736963 NZ_GG657909.1: 792 ZP_04567444.1 1153 Fusobacterium 489336 . . . 490343 mortiferum ATCC 9817 157736754 complement 793 YP_001489437.1 1154 Arcobacter (NC_009850.1: butzleri 500921 . . . 501916) RM4018 257125122 NC_013192.1: 794 YP_003163236.1 1155 Leptotrichia 327731 . . . 328735 buccalis C-1013-b 260891157 NZ_ACVB02000026.1: 795 ZP_05902420.1 1156 Leptotrichia 170989 . . . 171993 hofstadii F0254 262037878 complement 796 ZP_06011308.1 1157 Leptotrichia (NZ_ADAD01000064.1: goodfellowii 9188 . . . 10195) F0264 229859891 NZ_ABUT01000004.1: 797 ZP_04479548.1 1158 Streptobacillus 63190 . . . 64215 moniliformis DSM 12112 227371784 NZ_ABVB01000002.1: 798 ZP_03855267.1 1159 Veillonella 260421 . . . 261419 parvula DSM 2008 227498373 complement 799 ZP_03928523.1 1160 Acidaminococcus (NZ_ACGB01000001.1: sp. D21 109630 . . . 110643) 42525561 NC_002967.9: 800 NP_970659.1 1161 Treponema 48816 . . . 49823 denticola ATCC 35405 257456313 NZ_ACYH01000011.1: 801 ZP_05621510.1 1162 Treponema 210840 . . . 211847 vincentii ATCC 35580 15639088 NC_000919.1: 802 NP_218534.1 1163 Treponema 102879 . . . 103889 pallidum subsp. pallidum str. Nichols 228000316 complement 803 ZP_04047318.1 1164 Brachyspira (NZ_ABTG01000001.1: murdochii 1179041 . . . 1180048) DSM 12563 225619252 NC_012225.1: 804 YP_002720478.1 1165 Brachyspira 340269 . . . 341276 hyodysenteriae WA1 218960931 complement 805 YP_001740706.1 1166 Candidatus (NS_000195.1: Cloacamonas 716420. . . 717424) acidaminovorans 239878221 complement 806 EER05013.1 1167 Perkinsus (join(GG681098.1: marinus 49679 . . . 49966, ATCC 50983 GG681098.1: 50017 . . . 50325, GG681098.1: 50380 . . . 50442, GG681098.1: 50494 . . . 50605, GG681098.1: 50656 . . . 50780, GG681098.1: 50826 . . . 50908, GG681098.1: 50958 . . . 51039)) 119953373 NC_008710.1: 807 YP_945582.1 1168 Borrelia 614125 . . . 615171 turicatae 91E135 187918450 NC_010673.1: 808 YP_001884013.1 1169 Borrelia 616784 . . . 617842 hermsii DAH 203284493 NC_011229.1: 809 YP_002222233.1 1170 Borrelia 622676 . . . 623746 duttonii Ly 224534734 complement 810 ZP_03675306.1 1171 Borrelia (NZ_ABKB02000009.1: spielmanii 27640 . . . 28677) A14S 216263399 NZ_ABCU02000001.1: 811 ZP_03435394.1 1172 Borrelia 172066 . . . 173103 afzelii ACA-1 219685198 NZ_ABPZ02000001.1: 812 ZP_03540018.1 1173 Borrelia 172004 . . . 173041 garinii Far04 224532296 NZ_ABCY02000001.1: 813 ZP_03672928.1 1174 Borrelia 609419 . . . 610456 valaisiana VS116 15594934 NC_001318.1: 814 NP_212723.1 1175 Borrelia 608020 . . . 609078 burgdorferi B31 189485346 NS_000191.1: 815 YP_001956287.1 1176 uncultured 518918 . . . 519919 Termite group 1 bacterium phylotype Rs-D17 42560817 NC_005364.2: 816 NP_975268.1 1177 Mycoplasma 308545 . . . 309513 mycoides subsp. mycoides SC str. PG1 83319483 NC_007633.1: 817 YP_424216.1 1178 Mycoplasma 277239 . . . 278207 capricolum subsp. capricolum ATCC 27343 50364858 NC_006055.1: 818 YP_053283.1 1179 Mesoplasma 58892 . . . 59860 florum L1 110005214 complement 819 CAK99540.1 1180 Spiroplasma (AM285317.1: citri 14153 . . . 15130) 12045155 complement 820 NP_072966.1 1181 Mycoplasma (NC_000908.2: genitalium 368733 . . . 369695) G37 13508167 complement 821 NP_110116.1 1182 Mycoplasma (NC_000912.1: pneumoniae 515605 . . . 516567) M129 31544825 complement 822 NP_853403.1 1183 Mycoplasma (NC_004829.1: gallisepticum 851083 . . . 852075) R 26553955 complement 823 NP_757889.1 1184 Mycoplasma (NC_004432.1: penetrans 640803 . . . 641777) HF-2 54020554 complement 824 YP_116016.1 1185 Mycoplasma (NC_006360.1: hyopneumoniae 638554 . . . 639507) 232 240047219 NC_012806.1: 825 YP_002960607.1 1186 Mycoplasma 88435 . . . 89406 conjunctivae 148377406 NC_009497.1: 826 YP_001256282.1 1187 Mycoplasma 159649 . . . 160605 agalactiae PG2 238809713 complement 827 BAH69503.1 1188 Mycoplasma (AP009608.1: fermentans 242111 . . . 243064) PG18 71894663 complement 828 YP_278771.1 1189 Mycoplasma (NC_007294.1: synoviae 757812 . . . 758771) 53 15828708 NC_002771.1: 829 NP_326068.1 1190 Mycoplasma 274992 . . . 275948 pulmonis UAB CTIP 47459003 NC_006908.1: 830 YP_015865.1 1191 Mycoplasma 230100 . . . 231068 mobile 163K 148377754 NC_009497.1: 831 YP_001256630.1 1192 Mycoplasma 572993 . . . 573967 agalactiae PG2 116515056 NC_008513.1: 832 YP_802685.1 1193 Buchnera 131608 . . . 132594 aphidicola str. Cc (Cinara cedri) 187934490 NC_010674.1: 833 YP_001885432.1 1194 Clostridium 1263289 . . . 1264287 botulinum B str. Eklund 17B 150016048 NC_009617.1: 834 YP_001308302.1 1195 Clostridium 1384403 . . . 1385404 beijerinckii NCIMB 8052 254519224 complement 835 ZP_05131280.1 1196 Clostridium (NZ_EQ999773.1: sp. 7_2_43FAA 2015491 . . . 2016492) 182417251 NZ_ABDT01000035.2: 836 ZP_02948604.1 1197 Clostridium 9769 . . . 10770 butyricum 5521 18310707 complement 837 NP_562641.1 1198 Clostridium (NC_003366.1: perfringens 2001712 . . . 2002719) str. 13 255524273 complement 838 ZP_05391232.1 1199 Clostridium (NZ_ACVI01000014.1: carboxidivorans 63543 . . . 64547) P7 153954015 NC_009706.1: 839 YP_001394780.1 1200 Clostridium 1428554 . . . 1429555 kluyveri DSM 555 187778946 NZ_ABKW02000004.1: 840 ZP_02995419.1 1201 Clostridium 733017 . . . 734015 sporogenes ATCC 15579 28210926 NC_004557.1: 841 NP_781870.1 1202 Clostridium 1326340 . . . 1327359 tetani E88 253681395 NZ_ACSJ01000007.1: 842 ZP_04862192.1 1203 Clostridium 344343 . . . 345338 botulinum D str. 1873 118444574 complement 843 YP_878298.1 1204 Clostridium (NC_008593.1: novyi 1416375 . . . 1417373) NT 242260238 NZ_ACPD01000011.1: 844 ZP_04804960.1 1205 Clostridium 83320 . . . 84318 cellulovorans 743B 15895019 NC_003030.1: 845 NP_348368.1 1206 Clostridium 1890289 . . . 1891290 acetobutylicum ATCC 824 169247670 EU313773.1: 846 ACA51668.1 1207 Thermoanaerobacterium 40 . . . 1026 saccharolyticum 255257449 NZ_ACVG01000034.1: 847 ZP_05336886.1 1208 Thermoanaerobacterium 8635 . . . 9621 thermosaccharolyticum DSM 571 20807926 complement 848 NP_623097.1 1209 Thermoanaerobacter (NC_003869.1: tengcongensis 1451520 . . . 1452515) MB4 167040369 complement 849 YP_001663354.1 1210 Thermoanaerobacter (NC_010320.1: sp. X514 1738259 . . . 1739257) 220931863 NC_011899.1: 850 YP_002508771.1 1211 Halothermothrix 1110901 . . . 1111899 orenii H 168 258514457 complement 851 YP_003190679.1 1212 Desulfotomaculum (NC_013216.1: acetoxidans 1194895 . . . 1195899) DSM 771 188586231 complement 852 YP_001917776.1 1213 Natranaerobius (NC_010718.1: thermophilus 1692944 . . . 1693942) JW/NM-WN-LF 78044760 complement 853 YP_360288.1 1214 Carboxydothermus (NC_007503.1: hydrogenoformans 1302969 . . . 1303973) Z-2901 262295620 complement 854 EEY83551.1 1215 Bacteroides (GG705150.1: sp. 2_1_33B 648642 . . .649655) 154494088 complement 855 ZP_02033408.1 1216 Parabacteroides (NZ_AAXE02000107.1: merdae 241237 . . . 242250) ATCC 43184 34540818 complement 856 NP_905297.1 1217 Porphyromonas (NC_002950.2: gingivalis 1149763 . . . 1150773) W83 228471187 complement 857 ZP_04056000.1 1218 Porphyromonas (NZ_ACLR01000214.1: uenonis 23231 . . . 24238) 60-3 229496164 NZ_ACNN01000020.1: 858 ZP_04389884.1 1219 Porphyromonas 205218 . . . 206225 endodontalis ATCC 35406 160887812 complement 859 ZP_02068815.1 1220 Bacteroides (NZ_AAY1302000031.1: uniformis 6367 . . . 7386) ATCC 8492 218131945 NZ_ABVO01000052.1: 860 ZP_03460749.1 1221 Bacteroides 25694 . . . 26710 eggerthii DSM 20697 224536405 complement 861 ZP_03676944.1 1222 Bacteroides (NZ_ACCH01000118.1: cellulosilyticus 1796 . . . 2812) DSM 14838 53711769 NC_006347.1: 862 YP_097761.1 1223 Bacteroides 557297 . . . 558316 fragilis YCH46 237715344 complement 863 ZP_04545825.1 1224 Bacteroides (NZ_EQ973249.1: sp. D1 217217 . . . 218236) 224025178 NZ_ACBW01000140.1: 864 ZP_03643544.1 1225 Bacteroides 3350 . . . 4369 coprophilus DSM 18228 198274546 NZ_ABQC02000011.1: 865 ZP_03207078.1 1226 Bacteroides 44269 . . . 45279 plebeius DSM 17135 150003111 NC_009614.1: 866 YP_001297855.1 1227 Bacteroides 740818 . . . 741831 vulgatus ATCC 8482 258649233 complement 867 ZP_05736702.1 1228 Prevotella (NZ_ACIJ02000031.1: tannerae 14596 . . . 15612) ATCC 51259 261881160 NZ_ACKS01000109.1: 868 ZP_06007587.1 1229 Prevotella 4227 . . . 5276 bergensis DSM 17361 260593477 NZ_ACVA01000073.1: 869 ZP_05858935.1 1230 Prevotella 31053 . . . 32099 veroralis F0319 260910323 complement 870 ZP_05916997.1 1231 Prevotella (NZ_ACZS01000043.1: sp. oral taxon 472 34220 . . . 35257) str. F0295 212550465 complement 871 YP_002308782.1 1232 Candidatus (NC_011565.1: Azobacteroides 126538 . . . 127539) pseudotrichonymphae genomovar. CFP2 114566305 NC_008346.1: 872 YP_753459.1 1233 Syntrophomonas 872558 . . . 873550 wolfei subsp. wolfei str. Goettingen 139437229 NZ_AAVN02000001.1: 873 ZP_01771389.1 1234 Collinsella 368246 . . . 369226 aerofaciens ATCC 25986 210631306 complement 874 ZP_03296849.1 1235 Collinsella (NZ_ABXJ01000041.1: stercoris 3328 . . . 4308) DSM 13279 229814970 complement 875 ZP_04445308.1 1236 Collinsella (NZ_ABXH02000002.1: intestinalis 65772 . . . 66770) DSM 13280 221194458 complement 876 ZP_03567515.1 1237 Atopobium (NZ_ACFE01000001.1: rimae 86128 . . . 87273) ATCC 49626 257784450 complement 877 YP_003179667.1 1238 Atopobium (NC_013203.1: parvulum 723329 . . . 724309) DSM 20469 227516084 complement 878 ZP_03946133.1 1239 Atopobium (NZ_ACGK01000007.1: vaginae 63717 . . . 64691 DSM 15829 227872296 NZ_ACKX01000061.1: 879 ZP_03990654.1 1240 Oribacterium 10209 . . . 11261 sinus F0268 229824780 NZ_ACIN02000002.1: 880 ZP_04450849.1 1241 Abiotrophia 126870 . . . 127931 defectiva ATCC 49176 260443831 NZ_ACIQ01000073.1: 881 ZP_05797601.1 1242 Oribacterium 32192 . . . 33196 sp. oral taxon 078 str. F0262 225176688 complement 882 ZP_03730247.1 1243 Clostridium (NZ_ACFX01000006.1: sp. M62/1 113088 . . . 114089) 253578981 complement 883 ZP_04856252.1 1244 Ruminococcus (NZ_GG696046.1: sp. 5_1_39BFAA 364564 . . . 365595) 153813664 NZ_AAVO02000036.1: 884 ZP_01966332.1 1245 Ruminococcus 4823 . . . 5992 obeum ATCC 29174 255281061 complement 885 ZP_05345616.1 1246 Bryantella (NZ_ACCL02000005.1: formatexigens 162813 . . . 163811) DSM 14469 225571965 NZ_ACBZ01000008.1: 886 ZP_03780829.1 1247 Blautia 1408 . . . 2442 hydrogenotrophica DSM 10507 210612569 NZ_ABWO01000095.2: 887 ZP_03289360.1 1248 Clostridium 3132 . . . 4127 nexile DSM 1787 154505354 complement 888 ZP_02042092.1 1249 Ruminococcus (NZ_AAYG02000022.1: gnavus 50151 . . . 51146) ATCC 29149 197303064 NZ_ABOU02000039.1: 889 ZP_03168112.1 1250 Ruminococcus 71843 . . . 72838 lactaris ATCC 29176 153816169 complement 890 ZP_01968837.1 1251 Ruminococcus (NZ_AAVP02000015.1: torques 36559 . . . 37554) ATCC 27756 167758299 complement 891 ZP_02430426.1 1252 Clostridium (NZ_ABFY02000009.1: scindens 238358 . . . 239380) ATCC 35704 225570721 NZ_ABYI02000032.1: 892 ZP_03779744.1 1253 Clostridium 3477 . . . 4499 hylemonae DSM 15053 166031766 NZ_AAXA02000013.1: 893 ZP_02234595.1 1254 Dorea 54410 . . . 55414 formicigenerans ATCC 27755 153853264 complement 894 ZP_01994673.1 1255 Dorea (NZ_AAXB02000002.1: longicatena 216862 . . . 217857) DSM 13814 160879474 NC_010001.1: 895 YP_01558442.1 1256 Clostridium 1657582 . . . 1658577 phytofermentans ISDg 239624054 complement 896 ZP_04667085.1 1257 Clostridiales (NZ_DS990263.1: bacterium 658578 . . . 659573) 1_7_47FAA 160938034 complement 897 ZP_02085391.1 1258 Clostridium (NZ_ABCC02000027.1: bolteae 52316 . . . 53311) ATCC BAA-613 260437037 complement 898 ZP_05790853.1 1259 Butyrivibrio (NZ_ABWN01000017.1: crossotus 33488 . . . 34483) DSM 2876 154483586 complement 899 ZP_02026034.1 1260 Eubacterium (NZ_AAVL02000033.1: ventriosum 74910 . . . 75941) ATCC 27560 238916996 complement 900 YP_002930513.1 1261 Eubacterium (NC_012778.1: eligens 1076225 . . . 1077220) ATCC 27750 242309058 NZ_DS990446.1: 901 ZP_04808213.1 1262 Helicobacter 108718 . . . 109737 pullorum MIT 98-5489 224418114 complement 902 ZP_03656120.1 1263 Helicobacter (NZ_ABQS01000024.1: canadensis 18744 . . . 19745) MIT 98-5491 237752737 NZ_GG661974.1: 903 ZP_04583217.1 1264 Helicobacter 463241 . . . 464236 winghamensis ATCC BAA-430 32266808 complement 904 NP_860840.1 1265 Helicobacter (NC_004917.1: hepaticus 1266998 . . . 1267993) ATCC 51449 224436915 complement 905 ZP_03657896.1 1266 Helicobacter (NZ_ABQT01000013.1: cinaedi 10506 . . . 11522) CCUG 18818 167745652 complement 906 ZP_02417779.1 1267 Anaerostipes (NZ_ABAX03000002.1: caccae 101957 . . . 102961) DSM 14662 167765558 complement 907 ZP_02437622.1 1268 Clostridium (NZ_ABGC03000004.1: sp. SS2/1 50807 . . . 51820) 163814038 NZ_ABEY02000003.1: 908 ZP_02205430.1 1269 Coprococcus 15727 . . . 16794 eutactus ATCC 27759 168334441 complement 909 ZP_02692616.1 1270 Epulopiscium (NZ_ABEQ01000029.2: sp. ‘N.t. 21420 . . . 22418) morphotype B’ 257791476 NC_013204.1: 910 YP_003182082.1 1271 Eggerthella 2035882 . . . 2036880 lenta DSM 2243 256827068 complement 911 YP_003151027.1 1272 Cryptobacterium (NC_013170.1: curtum 735166 . . . 736167) DSM 15641 257063929 complement 912 YP_003143601.1 1273 Slackia (NC_013165.1: heliotrinireducens 1407526 . . . 1408521) DSM 20476 256757417 complement 913 ZP_05498135.1 1274 Clostridium (NZ_ACXX01000078.1: papyrosolvens 7706 . . . 8701) DSM 2782 196254011 NZ_ABVG01000076.1: 914 ZP_03152606.1 1275 Clostridium 9016 . . . 10092 thermocellum JW20 146297046 complement 915 YP_001180817.1 1276 Caldicellulosiruptor (NC_009437.1: saccharolyticus 2185773 . . . 2186804) DSM 8903 349833 L23147.1: 916 AAA72041.1 1277 Methanosarcina 207 . . . 1208 thermophila 20092407 complement 917 NP_618482.1 1278 Methanosarcina (NC_003552.1: acetivorans 4448053 . . . 4449054) C2A 73669327 complement 918 YP_305342.1 1279 Methanosarcina (NC_007355.1: barkeri 2275987 . . . 2276988) str. Fusaro 163734840 NZ_ABIG01000010.1: 919 ZP_02142278.1 1280 Roseobacter 132890 . . . 133867 litoralis Och 149 110678177 complement 920 YP_681184.1 1281 Roseobacter (NC_008209.1: denitrificans 803709 . . . 804707) OCh 114 159044374 complement 921 YP_001533168.1 1282 Dinoroseobacter (NC_009952.1: shibae 1904769 . . . 1905794) DFL 12 254512869 NZ_DS999532.1: 922 ZP_05124935.1 1283 Rhodobacteraceae 435221 . . . 436255 bacterium KLH11 260432366 complement 923 ZP_05786337.1 1284 Silicibacter (NZ_GG704596.1: lacuscaerulensis 1443685 . . . 1444707) ITI-1157 150376990 NC_009620.1: 924 YP_001313586.1 1285 Sinorhizobium 1371590 . . . 1372624 medicae WSM419 16264720 NC_003078.1: 925 NP_437512.1 1286 Sinorhizobium 1058537 . . . 1059529 meliloti 1021 239833801 complement 926 ZP_04682129.1 1287 Ochrobactrum (NZ_ACQA01000002.1: intermedium 595918 . . . 596886) LMG 3301 153010822 complement 927 YP_001372036.1 1288 Ochrobactrum (NC_00968.1: anthropi 862920 . . . 863897) ATCC 49188 187919084 complement 928 YP_001888115.1 1289 Burkholderia (NC_010676.1: phytofirmans 423371 . . . 424405) PsJN 91779405 NC_007952.1: 929 YP_554613.1 1290 Burkholderia 2605754 . . . 2606791 xenovorans LB400 186470979 NC_010625.1: 930 YP_001862297.1 1291 Burkholderia 763673 . . . 764704 phymatum STM815 73537607 complement 931 YP_297974.1 1292 Ralstonia (NC_007348.1: eutropha 372720 . . . 373757) JMP134 194292312 NC_010530.1: 932 YP_002008219.1 1293 Cupriavidus 1692071 . . . 1693105 taiwanensis 161521061 NC_010086.1: 933 YP_001584488.1 1294 Burkholderia 1600765 . . . 1601856 multivorans ATCC 17616 206563034 complement 934 YP_002233797.1 1295 Burkholderia (NC_011001.1: cenocepacia 1288493 . . . 1289527) J2315 90412230 complement 935 ZP_01220235.1 1296 Photobacterium (NZ_AAPH01000013.1: profundum 616 . . . 1653) 3TCK 224825256 complement 936 ZP_03698361.1 1297 Lutiella (NZ_ACIS01000005.1: nitroferrum 7593 . . . 8612) 2002 148973982 complement 937 ZP_01811515.1 1298 Vibrionales (NZ_AAZW01000001.1: bacterium 115872 . . . 116945) SWAT-3 84385317 complement 938 ZP_00988349.1 1299 Vibrio (NZ_AAMR01000001.1: splendidus 227808 . . . 228881) 12B01 149187938 NZ_ABCH01000003.1: 939 ZP_01866234.1 1300 Vibrio 163877 . . . 164926 shilonii AK1 260776268 complement 940 ZP_05885163.1 1301 Vibrio (NZ_ACZN01000015.1: coralliilyticus 316917 . . . 317966) ATCC BAA-450 45862014 AY498613.1: 941 AAS78789.1 1302 Paracoccus 8897 . . . 9853 denitrificans 77404622 NC_007488.1: 942 YP_345196.1 1303 Rhodobacter 38175 . . . 39173 sphaeroides 2.4.1 23630309 AY134843.1: 943 AAN08490.1 1304 Castellaniella 2180 . . . 3148 defragrans 83952615 NZ_AALY01000004.1: 944 ZP_00961345.1 1305 Roseovarius 6833 . . . 7819 nubinhibens ISM 56698382 complement 945 YP_168755.1 1306 Ruegeria (NC_003911.11: pomeroyi 3772593 . . . 3773606) DSS-3 149912659 NZ_ABCR01000001.1: 946 ZP_01901193.1 1307 Roseobacter 262172 . . . 263161 sp. AzwK-3b 126736835 complement 947 ZP_01752570.1 1308 Roseobacter (NZ_AAYC01000001.1: sp. SK209-2-6 23010 . . . 24029) 163732628 complement 948 ZP_02140073.1 1309 Roseobacter (NZ_ABIG01000003.1: litoralis 152433 . . . 153374) Och 149 89055338 NC_007802.1: 949 YP_510789.1 1310 Jannaschia 2864232 . . . 2865239 sp. CCS1 254459737 NZ_DS995276.1: 950 ZP_05073153.1 1311 Rhodobacterales 713420 . . . 714472 bacterium HTCC2083 116620211 NC_008536.1: 951 YP_822367.1 1312 Candidatus 1336713 . . . 1337708 Solibacter usitatus Ellin6076 95930364 NZ_AAEW02000012.1: 952 ZP_01313101.1 1313 Desulfuromonas 74280 . . . 75281 acetoxidans DSM 684 77920135 NC_007498.2: 953 YP_357950.1 1314 Pelobacter 2984046 . . . 2985047 carbinolicus DSM 2380 222054722 complement 954 YP_002537084.1 1315 Geobacter (NC_011979.1: sp. FRC-32 1793784 . . . 1794785) 148265418 NC_009483.1: 955 YP_001232124.1 1316 Geobacter 3992460 . . . 2993461 uraniireducens Rf4 39997800 NC_002939.4: 956 NP_953751.1 1317 Geobacter 2984470 . . . 2985471 sulfurreducens PCA 78222253 complement 957 YP_384000.1 1318 Geobacter (NC_007517.1: metallireducens 1150703 . . . 1151704) GS-15 118579718 complement 958 YP_900968.1 1319 Pelobacter (NC_008609.1: propionicus 1359173 . . . 1360177) DSM 2379 189424275 complement 959 YP_001951452.1 1320 Geobacter (NC_010814.1: lovleyi 1256052 . . . 1257053) SZ 255059775 NZ_ACPJ01000030.1: 960 ZP_05311922.1 1321 Geobacter 50425 . . . 51426 sp. M18 253700569 complement 961 YP_003021758.1 1322 Geobacter (NC_012918.1: sp. M21 2257017 . . . 2258018) 77920440 complement 962 YP_358255.1 1323 Pelobacter (NC_007498.2: carbinolicus 3332135 . . . 3333136) DSM 2380 227423754 NZ_ABTN01000011.1: 963 ZP_03906856.1 1324 Denitrovibrio 41883 . . . 42872 acetiphilus DSM 12809 193215894 NC_011026.1: 964 YP_001997093.1 1325 Chloroherpeton 2629909 . . . 2630916 thalassium ATCC 35110 150386298 complement 965 ZP_01924858.1 1326 Victivallis (NZ_ABDE01000122.1: vadensis 6609 . . . 7619) ATCC BAA-548 217034411 NZ_ABSX01000018.1: 966 ZP_03439825.1 1327 Helicobacter 29972 . . . 31570 pylori 98-10 254779508 complement 967 YP_003057614.1 1328 Helicobacter (NC_012973.1: pylori 876982 . . . 878538) B38 188527730 complement 968 YP_001910417.1 1329 Helicobacter (NC_010698.2: pylori 936737 . . . 938293) Shi470 15611908 complement 969 NP_223559.1 1330 Helicobacter (NC_000921.1: pylori 920263 . . . 921822) J99 109947805 complement 970 YP_665033.1 1331 Helicobacter (NC_008229.1: acinonychis 1105678 . . . 1107201) str. Sheeba 148926656 NZ_AASY01000008.1: 971 ZP_01810337.1 1332 Campylobacter 52243 . . . 53778 jejuni subsp. jejuni CG8486 57167700 complement 972 ZP_00366840.1 1333 Campylobacter (NZ_AAFL01000001.1: coli 232544 . . . 234046) RM2228 57242590 complement 973 ZP_00370527.1 1334 Campylobacter (NZ_AAFJ01000004.1: upsaliensis 6136 . . . 7638) RM3195 222823645 NC_012039.1: 974 YP_002575219.1 1335 Campylobacter 612447 . . . 613916 lari RM2100 154148075 complement 975 YP_001406718.1 1336 Campylobacter (NC_009714.1: hominis 1098161 . . . 1099597) ATCC BAA-381 257459711 NZ_ACYG01000019.1: 976 ZP_05624820.1 1337 Campylobacter 245785 . . . 247383 gracilis RM3268 118475502 complement 977 YP_891988.1 1338 Campylobacter (NC_008599.1: fetus subsp. 824533 . . . 825927) fetus 82-40 157164211 NC_009802.1: 978 YP_001466901.1 1339 Campylobacter 1056736 . . . 1058103 concisus 13826 154173700 complement 979 YP_001408221.1 1340 Campylobacter (NC_009715.1: curvus 947742 . . . 949112) 525.92 255322202 NZ_ACVQ01000017.1: 980 ZP_05363348.1 1341 Campylobacter 43696 . . . 45081 showae RM3277 225351910 NZ_ABXX02000003.1: 981 ZP_03742933.1 1342 Bifidobacterium 117844 . . . 119514 pseudocatenulatum DSM 20438 171743080 complement 982 ZP_02918887.1 1343 Bifidobacterium (NZ_ABIX02000002.1: dentium 2348529 . . . 2350229) ATCC 27678 154487476 complement 983 ZP_02028883.1 1344 Bifidobacterium (NZ_AAX1302000028.1: adolescentis 209751 . . . 211442) L2-32 229817818 complement 984 ZP_04448100.1 1345 Bifidobacterium (NZ_ABYS02000004.1: angulatum 899528 . . . 901234) DSM 20098 223467350 complement 985 ZP_03618886.1 1346 Bifidobacterium (NZ_ACCG01000014.1: breve 93417 . . . 95159) DSM 20213 227546035 NZ_ACHI01000009.1: 986 ZP_03976084.1 1347 Bifidobacterium 13043 . . . 14755 longum subsp. infantis ATCC 55813 213692597 NC_011593.1: 987 YP_002323183.1 1348 Bifidobacterium 1898224 . . . 1899936 longum subsp. infantis ATCC 15697 224282865 complement 988 ZP_03646187.1 1349 Bifidobacterium (NZ_ABQP01000009.1: bifidum 208984 . . . 210654) NCIMB 41171 227507562 NZ_ACGF01000124.1: 989 ZP_03937611.1 1350 Gardnerella 44384 . . . 46048 vaginalis ATCC 14019 183601499 complement 990 ZP_02962869.1 1351 Bifidobacterium (NZ_ABOT01000001.1: animalis subsp. 192971 . . . 194653) lactis HN019 261337301 complement 991 ZP_05965185.1 1352 Bifidobacterium (NZ_ABXB03000001.1: gallicum 137782 . . . 139455) DSM 20093 154507766 NZ_AAYI02000004.1: 992 ZP_02043408.1 1353 Actinomyces 231567 . . . 233045 odontolyticus ATCC 17982 227494860 complement 993 ZP_03925176.1 1354 Actinomyces (NZ_ACFG01000030.1: coleocanis 86700 . . . 88295) DSM 15436 19553946 complement 994 NP_601948.1 1355 Corynebacterium (NC_003450.3: glutamicum 2936506 . . . 2937891) ATCC 13032 25029147 complement 995 NP_739201.1 1356 Corynebacterium (NC_004369.1: efficiens 2758982 . . . 2760496) YS-314 38234612 complement 996 NP_940379.1 1357 Corynebacterium (NC_002935.2: diphtheriae 2103677 . . . 2105128) NCTC 13129 252124104 complement 997 ZP_04835255.1 1358 Corynebacterium (NZ_ACSH01000003.1: matruchotii 61905 . . . 63305) ATCC 14266 227489285 NZ_ABYP01000094.1: 998 ZP_03919601.1 1359 Corynebacterium 61648 . . . 63048 glucuronolyticum ATCC 51867 258561950 NZ_ACLJ01000070.1: 999 ZP_05708623.1 1360 Corynebacterium 13162 . . . 14523 genitalium ATCC 33030 227547861 NZ_ACHJ01000017.1: 1000 ZP_03977910.1 1361 Corynebacterium 9541 . . . 10899 lipophiloflavum DSM 44291 227502015 NZ_ACGD01000004.1: 1001 ZP_03932064.1 1362 Corynebacterium 82938 . . . 84305 accolens ATCC 49725 255325798 NZ_ACVP01000037.1: 1002 ZP_05366890.1 1363 Corynebacterium 5746 . . . 7107 tuberculostearicum SK141 227505901 NZ_ACGE01000122.1: 1003 ZP_03935950.1 1364 Corynebacterium 37468 . . . 38826 striatum ATCC 6940 227834110 complement 1004 YP_002835817.1 1365 Corynebacterum (NC_012590.1: aurimucosum 2492850 . . . 2494214) ATCC 700975 68535315 NC_007164.1: 1005 YP_250020.1 1366 Corynebacterium 307337 . . . 308848 jeikeium K411 172041418 complement 1006 YP_001801132.1 1367 Corynebacterium (NC_010545.1: urealyticum 2018026 . . . 2019369) DSM 7109 237786249 complement 1007 YP_002906954.1 1368 Corynebacterium (NC_012704.1: kroppenstedtii 1975731 . . . 1977287) DSM 44385 213965099 complement 1008 ZP_03393297.1 1369 Corynebacterium (NZ_ABZU01000003.1: amycolatum 128017 . . . 129543) SK46 225075788 complement 1009 ZP_03718987.1 1370 Neisseria (NZ_ACEN01000020.1: flavescens 5387 . . . 6889) NRL30031/H210 255067101 NZ_ACKO02000012.1: 1010 ZP_05318956.1 1371 Neisseria 64851 . . . 66353 sicca ATCC 29256 161869564 complement 1011 YP_001598731.1 1372 Neisseria (NC_010120.1: meningitidis 603202 . . . 604836) 053442 238022551 NZ_ACJW02000003.1: 1012 ZP_04602977.1 1373 Kingella 751672 . . . 753150 oralis ATCC 51147 83592714 complement 1013 YP_426466.1 1374 Rhodospirillum (NC_007643.1: rubrum 1625036 . . . 1626802) ATCC 11170 32490929 complement 1014 NP_871183.1 1375 Wigglesworthia (NC_004344.2: glossinidia 212680 . . . 214818) endosymbiont of Glossina brevipalpis 27904667 NC_004545.1: 1015 NP_777793.1 1376 Buchnera 186377 . . . 188524 aphidicola str. Bp (Baizongia pistaciae) 261415723 complement 1016 YP_003249406.1 1377 Fibrobacter (NC_013410.1: succinogenes subsp. 1639393 . . . 1640796) succinogenes S85 219556226 NZ_ABQH01000061.1: 1017 ZP_03535302.1 1378 Mycobacterium <3 . . . 1196 tuberculosis T17 228471665 complement 1018 ZP_04056438.1 1379 Capnocytophaga (NZ_ACLQ01000003.1: gingivalis 154599 . . . 155657) ATCC 33624 256370675 NC_013123.1: 1019 YP_003108500.1 1380 Candidatus 116952 . . . 117950 Sulcia muelleri SMDSEM 6685772 X89084.1: 1020 P77844 1381 Corynebacterium 1009 . . . 199 glutamicum 227876041 NZ_ACKW01000045.1: 1021 ZP_03994160.1 1382 Mobiluncus 33898 . . . 34887 mulieris ATCC 35243 227492324 NZ_ACCQ01000004.1: 1022 ZP_03922640.1 1383 Mobiluncus 214416 . . . 215417 curtisii ATCC 43063 225027017 NZ_ACEP01000064.1: 1023 ZP_03716209.1 1384 Eubacterium 10650 . . . 11609 halli DSM 3353 225028951 complement 1024 ZP_03718143.1 1385 Eubacterium (NZ_ACEP01000172.1: hallii 22364 . . . 23416) DSM 3353 257438679 complement 1025 ZP_05614434.1 1386 Faecalibacterium (NZ_ACOP02000029.1: prausnitzii 99 . . . 1124) A2-165 154496156 complement 1026 ZP_02034852.1 1387 Bacteroides (NZ_AAXG02000004.1: capillosus 103391 . . . 104380) ATCC 29799 225376322 NZ_ACFY01000086.1: 1027 ZP_03753543.1 1388 Roseburia 6940 . . . 7992 inulinivorans DSM 16841 257414121 complement 1028 ZP_04745275.2 1389 Roseburia (NZ_ABYJ02000202.1: intestinalis 41125 . . . 42165) L1-82 238923816 NC_012781.1: 1029 YP_002937332.1 1390 Eubacterium 1324280 . . . 1325263 rectale ATCC 33656 160893459 NZ_AAYW02000007.1: 1030 ZP_02074244.1 1391 Clostridium 63870 . . . 64919 sp. L2-50 229829305 complement 1031 ZP_04455374.1 1392 Shuttleworthia (NZ_ACIP02000002.1: satelles 495313 . . . 496299) DSM 14600 218282181 complement 1032 ZP_03488480.1 1393 Eubacterium (NZ_ABYT01000061.1: biforme 45 . . . 1016) DSM 3989 160916120 complement 1033 ZP_02078327.1 1394 Eubacterium (NZ_ABAW02000025.1: dolichum 71684 . . . 72694) DSM 3991 160915347 NZ_ABAW02000020.1: 1034 ZP_02077559.1 1395 Eubacterium 14646 . . . 15638 dolichum DSM 3991 212697404 NZ_ABXA01000047.1: 1035 ZP_03305532.1 1396 Anaerococcus 41715 . . . 42701 hydrogenalis DSM 7454 256545936 complement 1036 ZP_05473291.1 1397 Anaerococcus (NZ_ACXU01000022.1: vaginalis 22101 . . . 23087) ATCC 51170 227501001 NZ_ACGC01000115.1: 1037 ZP_03931050.1 1398 Anaerococcus 23122 . . . 24108 tetradius ATCC 35098 257067207 complement 1038 YP_003153463.1 1399 Anaerococcus (NC_013171.1: prevotii 1868769 . . . 1869752) DSM 20548 227485732 complement 1039 ZP_03916048.1 1400 Anaerococcus (NZ_ABYO01000196.1: lactolyticus 43814 . . . 44833) ATCC 51172 19746077 NC_003485.1: 1040 NP_607213.1 1401 Streptococcus 903788 . . . 904783 pyogenes MGAS8232 13622266 AE004092.1: 1041 AAK34003.1 1402 Streptococcus 923921 . . . 924916 pyogenes M1 GAS 222153008 NC_012004.1: 1042 YP_002562185.1 1403 Streptococcus 834034 . . . 835026 uberis 0140J 225868503 NC_012470.1: 1043 YP_002744451.1 1404 Streptococcus 1034662 . . . 1035663 equi subsp. Zooepidemicus 254997415 AP010655.1: 1044 BAH88016.1 1405 Streptococcus 1031526 . . . 1032521 mutans NN2025 171779341 NZ_ABJK02000020.1: 1045 ZP_02920305.1 1406 Streptococcus 38474 . . . 39472 infantarius subsp. infantarius ATCC BAA-102 76787123 complement 1046 YP_329798.1 1407 Streptococcus (NC_007432.1: agalactiae 1155758 . . . 1156750) A909 228477151 complement 1047 ZP_04061789.1 1408 Streptococcus (NZ_ACLO01000062.1: salivarius 54543 . . . 55526) SK126 55821439 complement 1048 YP_139881.1 1409 Streptococcus (NC_006448.1: thermophilus 1286014 . . . 1286997) LMG 18311 237650772 NZ_ABZC01000093.1: 1049 ZP_04525024.1 1410 Streptococcus 10653 . . . 11627 pneumoniae CCRI 1974 262282806 complement 1050 ZP_06060573.1 1411 Streptococcus (NZ_GG7049411.1: sp. 2_1_36FAA 11119 . . . 12096) 146318711 complement 1051 YP_001198423.1 1412 Streptococcus (NC_009442.1: suis 1032399 . . . 1033379) 05ZYH33 42518809 NC_005362.1: 1052 NP_964739.1 1413 Lactobacillus 788505 . . . 789482 johnsonii NCC 533 58337025 NC_006814.3: 1053 YP_193610.1 1414 Lactobacillus 698578 . . . 699567 acidophilus NCFM 227893214 NZ_ACGU01000037.1: 1054 ZP_04011019.1 1415 Lactobacillus 27358 . . . 28347 ultunensis DSM 16047 227877224 NZ_ACKR01000025.1: 1055 ZP_03995297.1 1416 Lactobacillus 14947 . . . 15936 crispatus JV-V01 260102516 NZ_ACLM01000112.1: 1056 ZP_05752753.1 1417 Lactobacillus 6924 . . . 7913 helveticus DSM 20075 227525975 NZ_ACGQ01000041.1: 1057 ZP_03956024.1 1418 Lactobacillus 59881 . . . 60858 jensenii JV-V16 228854857 complement 1058 ZP_04645187.1 1419 Lactobacillus (NZ_ACOY01000013.1: jensenii 251390 . . . 252367) 269-3 104773739 NC_008054.1: 1059 YP_618719.1 1420 Lactobacillus 547017 . . . 548006 delbrueckii subsp. bulgaricus ATCC 11842 259501464 NZ_ACLN01000013.1: 1060 ZP_05744366.1 1421 Lactobacillus 15438 . . . 16418 iners DSM 13335 16080818 complement 1061 NP_391646.1 1422 Bacillus (NC_000964.3: subtilis subsp. 3865355 . . . 3866326) subtilis str. 168 154687884 complement 1062 YP_001423045.1 1423 Bacillus (NC_009725.1: amyloliquefaciens 3590964 . . . 3591935) FZB42 52082282 complement 1063 YP_081073.1 1424 Bacillus (NC_006270.3: licheniformis 3821313 . . . 3822284) ATCC 14580 194016487 complement 1064 ZP_03055101.1 1425 Bacillus (NZ. ABRX01000004.1: pumilus 144981 . . . 145952) ATCC 7061 212640578 complement 1065 YP_002317098.1 1426 Anoxybacillus (NC_011567.1: flavithermus 2748264 . . . 2749247) WK1 239828646 complement 1066 YP_002951270.1 1427 Geobacillus (NC_012793.1: sp. WCH70 3393094 . . . 3394068) 138896990 complement 1067 YP_001127443.1 1428 Geobacillus (NC_009328.1: thermodenitrificans 3468960 . . . 3469934) NG80-2 56421950 complement 1068 YP_149268.1 1429 Geobacillus (NC_006510.1: kaustophilus 3456185 . . . 3457165) HTA426 149182788 NZ_ABCF01000043.1: 1069 ZP_01861251.1 1430 Bacillus 11583 . . . 12554 sp. SG-1 205375387 NZ_ABFU01000065.2: 1070 ZP_03228176.1 1431 Bacillus 12128 . . . 13099 coahuilensis m4-4 89101108 complement 1071 ZP_01173945.1 1432 Bacillus sp. (NZ_AAOX01000058.1: NRRL B-14911 10738 . . . 11715) 23100477 complement 1072 NP_693944.1 1433 Oceanobacillus (NC_004193.1: iheyensis 3134492 . . . 3135466) HTE831 229187615 complement 1073 ZP_04314753.1 1434 Bacillus (NZ_ACLU01000117.1: cereus 31676 . . . 32647) BGSC 6E1 46908338 complement 1074 YP_014727.1 1435 Listeria (NC_002973.6: monocytogenes 2171357 . . . 2172334) str. 4b F2365 229555968 NZ_ACCR01000020.1: 1075 ZP_04443757.1 1436 Listeria grayi 18426 . . . 19403 DSM 20601 15616385 complement 1076 NP_244690.1 1437 Bacillus (NC_002570.2: halodurans 3947889 . . . 3948881) C-125 56965668 complement 1077 YP_177402.1 1438 Bacillus (NC_006582.1: clausii 4069370 . . . 4070365) KSM-K16 229917170 complement 1078 YP_002885816.1 1439 Exiguobacterium (NC_012673.1: sp. AT1b 1410227 . . . 1411216) 172056261 NC_010556.1: 1079 YP_001812721.1 1440 Exiguobacterium 233988 . . . 234974 sibiricum 255-15 163762281 NZ_ABHZ01000002.1: 1080 ZP_02169346.1 1441 Bacillus 94480 . . . 95457 selenitireducens MLS10 242372812 NZ_ACJB01000048.1: 1081 ZP_04818386.1 1442 Staphylococcus 8122 . . . 9111 epidermidis M23864: W1 223042925 complement 1082 ZP_03612973.1 1443 Staphylococcus (NZ_ACFR01000002.1: capitis 330954 . . . 331943) SK14 239636796 complement 1083 ZP_04677798.1 1444 Staphylococcus (NZ_ACPZ01000027.1: warneri 569915 . . . 570904) L37603 27467277 NC_004461.1: 1084 NP_763914.1 1445 Staphylococcus 356818 . . . 357807 epidermidis ATCC 12228 258422775 NZ_ACKI01000006.1: 1085 ZP_05685678.1 1446 Staphylococcus 980 . . . 1966 aureus A9635 70727403 complement 1086 YP_254319.1 1447 Staphylococcus (NC_007168.1: haemolyticus 2403280 . . . 2404269) JCSC1435 228475091 NZ_ACLP01000011.1: 1087 ZP_04059818.1 1448 Staphylococcus 16037 . . . 17026 hominis SK119 150011041 EF456699.1: 1088 ABR57177.1 1449 Staphylococcus 1 . . . 987 xylosus 73663433 complement 1089 YP_302214.1 1450 Staphylococcus (NC_007356.1: saprophyticus subsp. 2190871 . . . 2191857) saprophyticus ATCC 15305 224475734 NC_012121.1: 1090 YP_002633340.1 1451 Staphylococcus 250258 . . . 251247 carnosus subsp. carnosus TM300 222152076 complement 1091 YP_002561236.1 1452 Macrococcus (NC_011999.1: caseolyticus 1968130 . . . 1969119) JCSC5402 227514417 NZ_ACGI01000058.1: 1092 ZP_03944466.1 1453 Lactobacillus 71225 . . . 72199 fermentum ATCC 14931 256848058 complement 1093 ZP_05553502.1 1454 Lactobacillus (NZ_GG698804.1: coleohominis 125094 . . . 126059) 101-4-CHN 227529580 NZ_ACGV01000117.1: 1094 ZP_03959629.1 1455 Lactobacillus 1634 . . . 2608 vaginalis ATCC 49540 148543634 NC_009513.1: 1095 YP_001271004.1 1456 Lactobacillus 451991 . . . 452965 reuteri DSM 20016 259502766 NZ_ACLL01000024.1: 1096 ZP_05745668.1 1457 Lactobacillus 18101 . . . 19072 antri DSM 16041 116618560 complement 1097 YP_818931.1 1458 Leuconostoc (NC_008531.1: mesenteroides subsp. 1461235 . . . 1462215) mesenteroides ATCC 8293 170016912 NC_010471.1: 1098 YP_001727831.1 1459 Leuconostoc 577967 . . . 578959 citreum KM20 241894748 complement 1099 ZP_04782044.1 1460 Weissella (NZ_ACKU01000002.1: paramesenteroides 15496 . . . 16476) ATCC 33313 118587037 NZ_AAUV01000054.1: 1100 ZP_01544468.1 1461 Oenococcus 44038 . . . 45132 oeni ATCC BAA-1163 259046893 complement 1101 ZP_05737294.1 1462 Granulicatella (NZ_ACKZ01000012.:1 adiacens 86436 . . . 87425) ATCC 49175 260584167 complement 1102 ZP_05851915.1 1463 Granulicatella (NZ_GG703805.1: elegans 786281 . . . 787264) ATCC 700633 163789527 complement 1103 ZP_02183965.1 1464 Carnobacterium (NZ_ABHH01000002.1: sp. AT7 8081 . . . 9061) 257870102 NZ_GG670288.1: 1104 ZP_05649755.1 1465 Enterococcus 145742 . . . 146725 gallinarum EG2 227517869 NZ_ACGL01000051.1: 1105 ZP_03947918.1 1466 Enterococcus 2376 . . . 3401 faecalis TX0104 227552175 complement 1106 ZP_03982224.1 1467 Enterococcus (NZ_ACHL01000118.1: faecium 1216 . . . 2232) TX1330 81428954 complement 1107 YP_395954.1 1468 Lactobacillus (NC_007576.1: sakei subsp. 1313600 . . . 1314586) sakei 23K 229823693 NZ_ACIL02000007.1: 1108 ZP_04449762.1 1469 Catonella 6499 . . . 7482 morbi ATCC 51271 125623617 NC_009004.1: 1109 YP_001032100.1 1470 Lactococcus 752099 . . . 753079 lactis subsp. cremoris MG1363 116494500 NC_008526.1: 1110 YP_806234.1 1471 Lactobacillus 981403 . . . 982380 casei ATCC 334 28377658 NC_004567.1: 1111 NP_784550.1 1472 Lactobacillus 748192 . . . 749169 plantarum WCFS1 116333321 NC_008497.1: 1112 YP_794848.1 1473 Lactobacillus 702374 . . . 703348 brevis ATCC 367 227524782 complement 1113 ZP_03954831.1 1474 Lactobacillus (NZ_ACGP01000192.1: hilgardii 2135 . . . 3112) ATCC 8290 11862872 AB035800.1: 1114 BAB19267.1 1475 Lactobacillus 1006 . . . 1992 sanfranciscensis 227528239 NZ_ACGS01000093.1: 1115 ZP_03958288.1 1476 Lactobacillus 64718 . . . 65695 ruminis ATCC 25644 90962126 complement 1116 YP_536042.1 1477 Lactobacillus (NC_007929.1: salivarius 1183945 . . . 1184922) UCC118 259504733 NZ_ACLK01000016.1: 1117 ZP_05747635.1 1478 Erysipelothrix 55937 . . . 56917 rhusiopathiae ATCC 19414 116492140 NC_008525.1: 1118 YP_803875.1 1479 Pediococcus 385259 . . . 386230 pentosaceus ATCC 25745 160946581 NZ_ABEE02000016.1: 1119 ZP_02093784.1 1480 Parvimonas 72101 . . . 73072 micra ATCC 33270 169825312 complement 1120 YP_001692923.1 1481 Finegoldia (NC_010376.1: magna 1782855 . . . 1783826) ATCC 29328 229542439 NZ_AAWV02000001.1: 1121 ZP_04431499.1 1482 Bacillus 1452854 . . . 1453825 coagulans 36D1 241888505 NZ_ACDZ02000004.1: 1122 ZP_04775813.1 1483 Gemella 11622 . . . 12602 haemolysans ATCC 10379

In addition, 201 phosphate acetyltransferase sequences that are characterized by two domains (DRTGG and PTA_PTB) are provided in Table 12b. MSA and phylogenetic analysis were performed as described above. Percent identity with respect to experimentally verified (or human curated) sequences is equal to or larger than 40, except for 4 sequences derived from plant organisms. Furthermore, hmmer search of the 201 sequences against the profile HMM of subfamily 2 (Table 14), clearly indicates that all Group 2 sequences belong to the PTA subfamily (least significant Evalue is 4.1e-93).

TABLE 12b SEQ ID NOs of phosphotransacetylase target gene coding regions and proteins GENBANK Nucleic GENBANK Amino Nucleotide Acid Amino Acid Acid GI Sequence SEQ Sequence SEQ Source Number Accession Information ID NO: Accession No. ID NO: Organism 152964825 complement 1484 YP_001360609.1 1685 Kineococcus (NC_009664.2: radiotolerans 1430885 . . . 1432984) SRS30216 88800302 complement 1485 ZP_01115869.1 1686 Reinekea (NZ_AAOE01000024.1: blandensis 59450 . . . 61609) MED297 254786809 complement 1486 YP_003074238.1 1687 Teredinibacter (NC_012997.1: turnerae 3139764 . . . 3141917) T7901 120554060 complement 1487 YP_958411.1 1688 Marinobacter (NC_008740.1: aquaeolei 1283732 . . . 1285885) VT8 83647145 NC_007645.1: 1488 YP_435580.1 1689 Hahella 4579211 . . . 4581358 chejuensis KCTC 2396 146308660 NC_009439.1: 1489 YP_001189125.1 1690 Pseudomonas 4021959 . . . 4024052 mendocina ymp 116048757 NC_008463.1: 1490 YP_792443.1 1691 Pseudomonas 4740071 . . . 4742185 aeruginosa UCBPP-PA14 28868382 complement 1491 NP_791001.1 1692 Pseudomonas (NC_004578.1: syringae pv. 1283902 . . . 1285992) tomato str. DC3000 70728320 complement 1492 YP_258069.1 1693 Pseudomonas (NC_004129.6: fluorescens 1081214 . . . 1083313) Pf-5 104780139 complement 1493 YP_606637.1 1694 Pseudomonas (NC_008027.1: entomophila 952666 . . . 954756) L48 226945506 complement 1494 YP_002800579.1 1695 Azotobacter (NC_012560.1: vinelandii 3530138 . . . 3532276) DJ 146281510 complement 1495 YP_001171663.1 1696 Pseudomonas (NC_009434.1: stutzeri 1238536 . . . 1240632) A1501 30248315 complement 1496 NP_840385.1 1697 Nitrosomonas (NC_004757.1: europaea 326321 . . . 328408) ATCC 19718 226946148 NC_012560.1: 1497 YP_002801221.1 1698 Azotobacter 4145609 . . . 4147684 vinelandii DJ 226357371 NC_012526.1: 1498 YP_002787111.1 1699 Deinococcus 2779899 . . . 2782016 deserti VCD115 94984159 complement 1499 YP_603523.1 1700 Deinococcus (NC_008025.1: geothermalis 46701 . . . 48812) DSM 11300 15805114 complement 1500 NP_293799.1 1701 Deinococcus (NC_001263.1: radiodurans 69707 . . . 71875) R1 89899079 complement 1501 YP_521550.1 1702 Rhodoferax (NC_007908.1: ferrireducens 264127 . . . 266178) T118 90422592 NC_007925.1: 1502 YP_530962.1 1703 Rhodopseudomonas 1181422 . . . 1183572 palustris BisB18 90423512 NC_007925.1: 1503 YP_531882.1 1704 Rhodopseudomonas 2183340 . . . 2185475 palustris BisB18 115525859 NC_008435.1: 1504 YP_782770.1 1705 Rhodopseudomonas 4320999 . . . 4323140 palustris BisA53 167574473 complement 1505 ZP_02367347.1 1706 Burkholderia (NZ_ABBG01000507.1: oklahomensis 7891 . . . 9969) C6786 83594327 complement 1506 YP_428079.1 1707 Rhodospirillum (NC_007643.1: rubrum 3449832 . . . 3451943) ATCC 11170 90422165 NC_007925.1: 1507 YP_530535.1 1708 Rhodopseudomonas 696325 . . . 698388 palustris BisB18 34496985 complement 1508 NP_901200.1 1709 Chromobacterium (NC_005085.1: violaceum 1636285 . . . 1638366) ATCC 12472 224825239 complement 1509 ZP_03698345.1 1710 Lutiella (NZ_ACIS01000004.1: nitroferrum 398128 . . . 400215) 2002 148652157 complement 1510 YP_001279250.1 1711 Psychrobacter (NC_009524.1: sp. PRwf-1 415573 . . . 417720) 93005047 complement 1511 YP_579484.1 1712 Psychrobacter (NC_007969.1: cryohalolentis 257926 . . . 260082) K5 257453691 NZ_ACYI01000010.1: 1512 ZP_05618978.1 1713 Enhydrobacter 16653 . . . 18797 aerosaccus SK60 255321153 NZ_ACVR01000080.1: 1513 ZP_05362319.1 1714 Acinetobacter 44385 . . . 46529 radioresistens SK82 50083778 complement 1514 YP_045288.1 1715 Acinetobacter (NC_005966.1: sp. ADP1 527524 . . . 529686) 260549093 NZ_GG704496.1: 1515 ZP_05823314.1 1716 Acinetobacter 86045 . . . 88189 sp. RUH2624 226953952 complement 1516 ZP_03824416.1 1717 Acinetobacter (NZ_ABYN01000201.1: sp. ATCC 27244 23157 . . . 25289) 153005955 NC_009675.1: 1517 YP_001380280.1 1718 Anaeromyxobacter 3624676 . . . 3626811 sp. Fw109-5 86159318 complement 1518 YP_466103.1 1719 Anaeromyxobacter (NC_007760.1: dehalogenans 3267950 . . . 3270094) 2CP-C 52425053 complement 1519 YP_088190.1 1720 Mannheimia (NC_006300.1: succiniciproducens 977458 . . . 979596) MBEL55E 152979320 NC_009655.1: 1520 YP_001344949.1 1721 Actinobacillus 1823344 . . . 1825485 succinogenes 130Z 251792685 NC_012913.1: 1521 YP_003007411.1 1722 Aggregatibacter 968721 . . . 970856 aphrophilus NJ8700 145633066 NZ_AAZF01000004.1: 1522 ZP_01788798.1 1723 Haemophilus 73469 . . . 75604 influenzae 3655 113460945 complement 1523 YP_719012.1 1724 Haemophilus (NC_008309.1: somnus 873911 . . . 876049) 129PT 15602570 NC_002663.1: 1524 NP_245642.1 1725 Pasteurella 821181 . . . 823319 multocida subsp. multocida str. Pm70 260913970 complement 1525 ZP_05920444.1 1726 Pasteurella (NZ_ACZR01000013.1: dagmatis 172766 . . . 174904) ATCC 43325 53729159 complement 1526 ZP_00133992.2 1727 Actinobacillus (NZ_AACK01000004.1: pleuropneumoniae 12180 . . . 14318) serovar 1 str. 4074 240949203 NZ_ACQL01000097.1: 1527 ZP_04753547.1 1728 Actinobacillus 15931 . . . 18069 minor NM305 33152520 NC_002940.2: 1528 NP_873873.1 1729 Haemophilus 1192390 . . . 1194528 ducreyi 35000HP 254362832 NZ_DS264681.1: 1529 ZP_04978908.1 1730 Mannheimia 4949 . . . 7084 haemolytica PHL213 219870647 NC_011852.1: 1530 YP_002475022.1 1731 Haemophilus 435431 . . . 437566 parasuis SH0165 258637834 NZ_ACYJ01000022.1: 1531 ZP_05730581.1 1732 Pantoea 41620 . . . 43764 sp. At-9b 188533336 complement 1532 YP_001907133.1 1733 Erwinia (NC_010694.1: tasmaniensis 1324250 . . . 1326379) Et1/99 85059585 NC_007712.1: 1533 YP_455287.1 1734 Sodalis 2759501 . . . 2761645 glossinidius str. ‘morsitans’ 258631105 complement 1534 ZP_05723922.1 1735 Dickeya (NZ_ACYK01000004.1: dadantii 104546 . . . 106687) Ech586 261820783 complement 1535 YP_003258889.1 1736 Pectobacterium (NC_013421.1: wasabiae 1606509 . . . 1608647) WPP163 242239978 NC_012880.1: 1536 YP_002988159.1 1737 Dickeya 3004583 . . . 3006724 dadantii Ech703 22125515 complement 1537 NP_668938.1 1738 Yersinia (NC_004088.1: pestis 1788905 . . . 1791058) KIM 10 157371554 NC_009832.1: 1538 YP_001479543.1 1739 Serratia 3673293 . . . 3675482 proteamaculans 568 238920583 NC_012779.1: 1539 YP_002934098.1 1740 Edwardsiella 2589897 . . . 2592035 ictaluri 93-146 197285630 NC_010554.1: 1540 YP_002151502.1 1741 Proteus 1898593 . . . 1900737 mirabilis HI4320 37526984 NC_005126.1: 1541 NP_930328.1 1742 Photorhabdus 3612456 . . . 3614597 luminescens subsp. laumondii TTO1 238895817 NC_012731.1: 1542 YP_002920553.1 1743 Klebsiella 3763302 . . . 3765449 pneumoniae NTUH-K2044 146312483 NC_009436.1: 1543 YP_001177557.1 1744 Enterobacter 3080629 . . . 3082770 sp. 638 260598715 NC_013282.1: 1544 YP_003211286.1 1745 Cronobacter 3057676 . . . 3059814 turicensis 601935 D21123.1: 1545 BAA04663.1 1746 Escherichia 77 . . . 2218 coli 238898722 complement 1546 YP_002924403.1 1747 Candidatus (NC_012751.1: Hamiltonella 1494526 . . . 1496655) defensa 5AT (Acyrthosiphon pisum) 227114079 NZ_ABVX01000029.1: 1547 ZP_03827735.1 1748 Pectobacterium 23117 . . . 25261 carotovorum subsp. brasiliensis PBR1692 89072717 complement 1548 ZP_01159282.1 1749 Photobacterium (NZ_AAOU01000004.1: sp. SKA34 98074 . . . 100221) 54309953 NC_006370.1: 1549 YP_130973.1 1750 Photobacterium 3245262 . . . 3247418 profundum SS9 262274670 NZ_ADAQ01000011.1: 1550 ZP_06052481.1 1751 Grimontia 496361 . . . 498520 hollisae CIP 101886 260768101 complement 1551 ZP_05877035.1 1752 Vibrio (NZ_ACZP01000013.1: furnissii 239301 . . . 241427) CIP 102972 260773044 NZ_ACZO01000006.1: 1552 ZP_05881960.1 1753 Vibrio 1066216 . . . 1068360 metschnikovii CIP 69.14 163802859 complement 1553 ZP_02196748.1 1754 Vibrio (NZ_ABGR01000013.1: sp. AND4 61871 . . . 64036) 37680318 NC_005139.1: 1554 NP_934927.1 1755 Vibrio 2144915 . . . 2147059 vulnificus YJ016 149188151 complement 1555 ZP_01866446.1 1756 Vibrio (NZ_ABCH01000004.1: shilonii 163878 . . . 166022) AK1 218708991 complement 1556 YP_002416612.1 1757 Vibrio (NC_011753.1: splendidus 1031606 . . . 1033810) LGP32 209695557 NC_011312.1: 1557 YP_002263486.1 1758 Aliivibrio 2262635 . . . 2264806 salmonicida LFI1238 229525709 complement 1558 ZP_04415114.1 1759 Vibrio (NZ_ACHV01000001.1: cholerae bv. 2574339 . . . 2576483) albensis VL426 145300284 NC_009348.1: 1559 YP_001143125.1 1760 Aeromonas 3681431 . . . 3683584 salmonicida subsp. salmonicida A449 237807651 complement 1560 YP_002892091.1 1761 Tolumonas (NC_012691.1: auensis 958413 . . . 960569) DSM 9187 90407162 complement 1561 ZP_01215350.1 1762 Psychromonas (NZ_AAPG01000006.1: sp. CNPT3 41954 . . . 44116) 119946918 NC_008709.1: 1562 YP_944598.1 1763 Psychromonas 4084304 . . . 4086466 ingrahamii 37 157374843 complement 1563 YP_001473443.1 1764 Shewanella (NC_009831.1: sediminis 2041698 . . . 2043839) HAW-EB3 170727231 NC_010506.1: 1564 YP_001761257.1 1765 Shewanella 3531467 . . . 3533608 woodyi ATCC 51908 127513322 NC_009092.1: 1565 YP_001094519.1 1766 Shewanella 2807561 . . . 2809699 loihica PV-4 167624517 NC_010334.1: 1566 YP_001674811.1 1767 Shewanella 3149368 . . . 3151515 halifaxensis HAW-EB4 117919999 complement 1567 YP_869191.1 1768 Shewanella (NC_008577.1: sp. ANA-3 1806421 . . . 180857 119774631 complement 1568 YP_927371.1 1769 Shewanella (NC_008700.1: amazonensis 1807689 . . . 1809827) SB2B 114563647 NC_008345.1: 1569 YP_751160.1 1770 Shewanella 2956515 . . . 2958662 frigidimarina NCIMB 400 91793762 NC_007954.1: 1570 YP_563413.1 1771 Shewanella 2868611 . . . 2870791 denitrificans OS217 157376672 NC_009831.1: 1571 YP_001475272.1 1772 Shewanella 4313346 . . . 4315484 sediminis HAW-EB3 167624655 complement 1572 YP_001674949.1 1773 Shewanella (NC_010334.1: halifaxensis 3320048 . . . 3322198) HAW-EB4 239996136 complement 1573 ZP_04716660.1 1774 Alteromonas (NZ_ABQB01000564.1: macleodii 6079 . . . 8301) ATCC 27126 109898905 NC_008228.1: 1574 YP_662160.1 1775 Pseudoalteromonas 3144369 . . . 3146513 atlantica T6c 119469286 NZ_AAVS01000006.1: 1575 ZP_01612225.1 1776 Alteromonadales 30053 . . . 32206 bacterium TW-7 88860001 complement 1576 ZP_01134640.1 1777 Pseudoalteromonas (NZ_AAOH01000005.1: tunicata 230650 . . . 232797) D2 71282469 NC_003910.7: 1577 YP_269873.1 1778 Colwellia 3309465 . . . 3311585 psychrerythraea 34H 152996332 NC_009654.1: 1578 YP_001341167.1 1779 Marinomonas 2608121 . . . 2610220 sp. MWYL1 87121463 NZ_AANE01000011.1: 1579 ZP_01077352.1 1780 Marinomonas 112021 . . . 114096 sp. MED121 146328905 NC_009446.1: 1580 YP_001209362.1 1781 Dichelobacter 489780 . . . 491837 nodosus VCS1703A 258544959 NZ_ACKY01000059.1: 1581 ZP_05705193.1 1782 Cardiobacterium 4448 . . . 6562 hominis ATCC 15826 262104765 complement 1582 EEY62817.1 1783 Phytophthora (DS028152.1: infestans 677306 . . . 679621) T30-4 262104764 complement 1583 EEY62816.1 1784 Phytophthora (DS028152.1: infestans 674496 . . . 676781) T30-4 159472743 XM_001694452.1: 1584 XP_001694504.1 1785 Chlamydomonas 258 . . . 2636 reinhardtii 168000833 XM_001753068.1: 1585 XP_001753120.1 1786 Physcomitrella 1 . . . 2367 patens subsp. Patens 172038009 complement 1586 YP_001804510.1 1787 Cyanothece (NC_010546.1: sp. ATCC 51142 3214848 . . . 3216944) 126658068 NZ_AAXW01000014.1: 1587 ZP_01729220.1 1788 Cyanothece 79066 . . . 81162 sp. CCY0110 257060449 NC_013161.1: 1588 YP_003138337.1 1789 Cyanothece 2659296 . . . 2661419 sp. PCC 8802 218441705 complement 1589 YP_002380034.1 1790 Cyanothece (NC_011729.1: sp. PCC 7424 5341705 . . . 5343810) 166368837 NC_010296.1: 1590 YP_001661110.1 1791 Microcystis 5646854 . . . 5648950 aeruginosa NIES-843 220909840 NC_011884.1: 1591 YP_002485151.1 1792 Cyanothece 4551169 . . . 4553265 sp. PCC 7425 16330299 NC_000911.1: 1592 NP_441027.1 1793 Synechocystis 1250442 . . . 1252535 sp. PCC 6803 86142732 NZ_AANC01000005.1: 1593 ZP_01061171.1 1794 Leeuwenhoekiella 209172 . . . 211268 blandensis MED217 146301271 complement 1594 YP_001195862.1 1795 Flavobacterium (NC_009441.1: johnsoniae 4208789 . . . 4210882) UW101 260061847 NC_013222.1: 1595 YP_003194927.1 1796 Robiginitalea 1408358 . . . 1410454 biformata HTCC2501 88713711 complement 1596 ZP_01107792.1 1797 Flavobacteriales (NZ_AAOC01000008.1: bacterium 22821 . . . 24917) HTCC2170 86133149 complement 1597 ZP_01051731.1 1798 Polaribacter (NZ_CH902588.1: sp. MED152 146636 . . . 148729) 88803680 NZ_AAOG01000005.1: 1598 ZP_01119204.1 1799 Polaribacter 54861 . . . 56951 irgensii 23-P 213962668 NZ_ABZV01000006.1: 1599 ZP_03390929.1 1800 Capnocytophaga 103363 . . . 105438 sputigena ATCC 33612 256820698 complement 1600 YP_003141977.1 1801 Capnocytophaga (NC_013162.1: ochracea 2243972 . . . 2246047) DSM 7271 46581432 NC_002937.3: 1601 YP_012240.1 1802 Desulfovibrio 3152216 . . . 3154330 vulgaris str. Hildenborough 218886955 NC_011769.1: 1602 YP_002436276.1 1803 Desulfovibrio 2286534 . . . 2288648 vulgaris str. ‘Miyazaki F’ 78358281 NC_007519.1: 1603 YP_389730.1 1804 Desulfovibrio 3235663 . . . 3237822 desulfuricans subsp. desulfuricans str. G20 242280036 complement 1604 YP_002992165.1 1805 Desulfovibrio (NC_012881.1: salexigens 2812652 . . . 2814769) DSM 2638 258405159 complement 1605 YP_003197901.1 1806 Desulfohalobium (NC_013223.1: retbaense 1218708 . . . 1220816) DSM 5692 256828849 NC_013173.1: 1606 YP_003157577.1 1807 Desulfomicrobium 1143375 . . . 1145477 baculatum DSM 4028 225198782 complement 1607 ZP_03737911.1 1808 Desulfonatronospira (NZ_ACJN01000010.1: thiodismutans 60728 . . . 62824) ASO3-1 242278203 NC_012881.1: 1608 YP_002990332.1 1809 Desulfovibrio 802309 . . . 804414 salexigens DSM 2638 212704109 complement 1609 ZP_03312237.1 1810 Desulfovibrio (NZ_ABXU01000065.1: piger 34368 . . . 36470) ATCC 29098 220903578 complement 1610 YP_002478890.1 1811 Desulfovibrio (NC_011883.1: desulfuricans 357004 . . . 359112) subsp. desulfuricans str. ATCC 27774 51244410 NC_006138.1: 1611 YP_064294.1 1812 Desulfotalea 608983 . . . 611115 psychrophila LSv54 94986723 NC_008011.1: 1612 YP_594656.1 1813 Lawsonia 347892 . . . 350012 intracellularis PHE/MN1-00 119488858 complement 1613 ZP_01621820.1 1814 Lyngbya (NZ_AAVU01000021.1: sp. PCC 8106 33384 . . . 35414) 209524350 NZ_ABYK01000010.1: 1614 ZP_03272899.1 1815 Arthrospira 35808 . . . 37916 maxima CS-328 116748909 NC_008554.1: 1615 YP_845596.1 1816 Syntrophobacter 1826816 . . . 1828915 fumaroxidans MPOB 241776655 NZ_ACQQ01000008.1: 1616 ZP_04773932.1 1817 Allochromatium 86078 . . . 88198 vinosum DSM 180 32476008 NC_005027.1: 1617 NP_869002.1 1818 Rhodopirellula 5198833 . . . 5200932 baltica SH 1 78776256 NC_007575.1: 1618 YP_392571.1 1819 Sulfurimonas 60204 . . . 62282 denitrificans DSM 1251 254458291 complement 1619 ZP_05071717.1 1820 Campylobacterales (NZ_DS995288.1: bacterium 173480 . . . 175561) GD 1 229532518 NZ_ABUV01000006.1: 1620 ZP_04421899.1 1821 Sulfurospirillum 73569 . . . 75677 deleyianum DSM 6946 152993574 NC_009663.1: 1621 YP_001359295.1 1822 Sulfurovum 2069625 . . . 2071724 sp. NBC37-1 120401715 NC_008726.1: 1622 YP_951544.1 1823 Mycobacterium 740616 . . . 742694 vanbaalenii PYR-1 145220810 complement 1623 YP_001131488.1 1824 Mycobacterium (NC_009338.1: gilvum 189392 . . . 191515) PYR-GCK 108797517 NC_008146.1: 1624 YP_637714.1 1825 Mycobacterium 594117 . . . 596231 sp. MCS 118473540 NC_008596.1: 1625 YP_885188.1 1826 Mycobacterium 867578 . . . 869656 smegmatis str. MC2 155 169631304 complement 1626 YP_001704953.1 1827 Mycobacterium (NC_010397.1: abscessus 4294451 . . . 4296532) 240168870 NZ_ACBV01000011.1: 1627 ZP_04747529.1 1828 Mycobacterium 33884 . . . 35974 kansasii ATCC 12478 183980733 NC_010612.1: 1628 YP_001849024.1 1829 Mycobacterium 853987 . . . 856071 marinum M 15607549 NC_000962.2: 1629 NP_214922.1 1830 Mycobacterium 491786 . . . 493858 tuberculosis H37Rv 41409983 NC_002944.2: 1630 NP_962819.1 1831 Mycobacterium 4345845 . . . 4347932 avium subsp. paratuberculosis K-10 254818871 complement 1631 ZP_05223872.1 1832 Mycobacterium (NZ_ABIN01000026.1: intracellulare 2280 . . . 4439) ATCC 13950 226304961 NC_012490.1: 1632 YP_002764919.1 1833 Rhodococcus 1605870 . . . 1607948 erythropolis PR4 111019190 complement 1633 YP_702162.1 1834 Rhodococcus (NC_008268.1: jostii 2308925 . . . 2311045) RHA1 54027320 complement 1634 YP_121562.1 1835 Nocardia (NC_006361.1: farcinica 5652808 . . . 5654895) IFM 10152 227978095 NZ_ABVA01000001.1: 1635 ZP_04025361.1 1836 Tsukamurella 785441 . . . 787570 paurometabola DSM 20162 262204223 complement 1636 YP_003275431.1 1837 Gordonia (NC_013441.1: bronchialis 4704088 . . . 4706208) DSM 43247 256831883 complement 1637 YP_003160610.1 1838 Jonesia (NC_013174.1: denitrificans 683848 . . . 685947) DSM 20603 260517199 complement 1638 ZP_05816650.1 1839 Sanguibacter (NZ_ABUN01000002.1: keddieii 90744 . . . 92939) DSM 10542 229243856 complement 1639 ZP_04368027.1 1840 Cellulomonas (NZ_ABTJ01000131.1: flavigena 4381 . . . 6468) DSM 20109 229821528 NC_012669.1: 1640 YP_002883054.1 1841 Beutenbergia 3401218 . . . 3403323 cavernae DSM 12333 227428424 complement 1641 ZP_03911481.1 1842 Xylanimonas (NZ_ABVC01000008.1: cellulosilytica 150308 . . . 152407) DSM 15894 119717178 complement 1642 YP_924143.1 1843 Nocardioides (NC_008699.1: sp. JS614 3139954 . . . 3142044) 227381337 complement 1643 ZP_03864789.1 1844 Kribbella (NZ_ABUC01000011.1: flavida 233655 . . . 235784) DSM 17836 88856399 NZ_AAOB01000010.1: 1644 ZP_01131057.1 1845 marine 2970 . . . 5138 actinobacterium PHSC20C1 170780609 NC_010407.1: 1645 YP_001708941.1 1846 Clavibacter 179995 . . . 182112 michiganensis subsp. Sepedonicus 50954174 NC_006087.1: 1646 YP_061462.1 1847 Leifsonia 335128 . . . 337257 xyli subsp. xyli str. CTCB07 114331961 complement 1647 YP_748183.1 1848 Nitrosomonas (NC_008344.1: eutropha 2107910 . . . 2110039) C91 256395328 complement 1648 YP_003116892.1 1849 Catenulispora (NC_013131.1: acidiphila 7130890 . . . 7133034) DSM 44928 258650827 NC_013235.1: 1649 YP_003199983.1 1850 Nakamurella 639398 . . . 641491 multipartita DSM 44233 257068066 complement 1650 YP_003154321.1 1851 Brachybacterium (NC_013172.1: faecium 995592 . . . 997667) DSM 4810 227497260 complement 1651 ZP_03927492.1 1852 Actinomyces (NZ_ACFH01000109.1: urogenitalis 3054 . . . 5108) DSM 15434 256824971 NC_013169.1: 1652 YP_003148931.1 1853 Kytococcus 1149994 . . . 1152081 sedentarius DSM 20547 260455562 NZ_ACZH01000022.1: 1653 ZP_05803950.1 1854 Streptomyces 9845 . . . 11917 flavogriseus ATCC 33331 182435904 NC_010572.1: 1654 YP_001823623.1 1855 Streptomyces 2506931 . . . 2509012 griseus subsp. griseus NBRC 13350 254387454 NZ_DS570624.1: 1655 ZP_05002693.1 1856 Streptomyces 26512 . . . 28596 clavuligerus ATCC 27064 254400535 NZ_DS570905.1: 1656 ZP_05015493.1 1857 Streptomyces 178100 . . . 180199 sviceus ATCC 29083 256813645 NZ_ACFA01000303.1: 1657 ZP_05538660.1 1858 Streptomyces 9482 . . . 11584 griseoflavus Tu4000 239928836 NZ_ABYA01000185.1: 1658 ZP_04685789.1 1859 Streptomyces 2180 . . . 4282 ghanaensis ATCC 14672 256804684 complement 1659 ZP_05534308.1 1860 Streptomyces (NZ_ACEZ01000169.1: viridochromogenes 8822 . . . 10924) DSM 40736 256785123 NZ_ACEY01000098.1: 1660 ZP_05523554.1 1861 Streptomyces 97187 . . . 99280 lividans TK24 29829365 NC_003155.4: 1661 NP_823999.1 1862 Streptomyces 3467325 . . . 3469415 avermitilis MA-4680 260646824 FN554889.1: 1662 CBG69921.1 1863 Streptomyces 3224239 . . . 3226344 scabiei 87.22 239982381 complement 1663 ZP_04704905.1 1864 Streptomyces (NZ_ABYC01000362.1: albus 40088 . . . 42163) J1074 254382385 complement 1664 ZP_04997745.1 1865 Streptomyces (NZ_DS570390.1: sp. Mg1 111950 . . . 114034) 256769973 complement 1665 ZP_05509147.1 1866 Streptomyces (NZ_ACEW01000403.1: sp. C 16454 . . . 18541) 256776255 NZ_ACEX01000277.1: 1666 ZP_05514718.1 1867 Streptomyces 2545 . . . 4641 hygroscopicus ATCC 53653 254378850 NZ_DS570550.1: 1667 ZP_04994290.1 1868 Streptomyces 40417 . . . 42507 sp. SPB74 229854086 complement 1668 ZP_04474082.1 1869 Streptosporangium (NZ_ABUU01000066.1: roseum 39364 . . . 41415) DSM 43021 145596204 NC_009380.1: 1669 YP_001160501.1 1870 Salinispora 4234932 . . . 4237007 tropica CNB-440 159039600 NC_009953.1: 1670 YP_001538853.1 1871 Salinispora 4631907 . . . 4633976 arenicola CNS-205 238060866 NZ_GG657738.1: 1671 ZP_04605575.1 1872 Micromonospora 2330097 . . . 2332163 sp. ATCC 39149 116671783 complement 1672 YP_832716.1 1873 Arthrobacter (NC_008541.1: sp. FB24 3648900 . . . 3651011) 148807608 complement 1673 ABR13603.1 1874 Arthrobacter (EF6018801: oxydans 72 . . . 2150) 239916738 complement 1674 YP_002956296.1 1875 Micrococcus (NC_012803.1: luteus 190981 . . . 193056) NCTC 2665 255326162 NZ_ACVO01000004.1: 1675 ZP_05367249.1 1876 Rothia 127571 . . . 129661 mucilaginosa ATCC 25296 184199797 complement 1676 YP_001854004.1 1877 Kocuria (NC_010617.1: rhizophila 164688 . . . 166781) DC2201 254368446 NZ_DS264133.1: 1677 ZP_04984463.1 1878 Francisella 69966 . . . 72062 tularensis subsp. holarctica FSC022 167626922 complement 1678 YP_001677422.1 1879 Francisella (NC_010336.1: philomiragia 741506 . . . 743602) subsp. philomiragia ATCC 25017 94676965 NC_007984.1: 1679 YP_588827.1 1880 Baumannia 392877 . . . 395012 cicadellinicola str. Hc (Homalodisca coagulata) P57273 BA000003.2: 1680 NP_240007.1 1881 Buchnera 189582 . . . 191708 aphidicola str. APS (Acyrthosiphon pisum) 254444018 NZ_DS990592.1: 1681 ZP_05057494.1 1882 Verrucomicrobiae 1298899 . . . 1201010 bacterium DG1235 171914782 NZ_ABIZ01000001.1: 1682 ZP_02930252.1 1883 Verrucomicrobium 6593044 . . . 6595128 spinosum DSM 4136 114777389 NZ_AATS01000006.1: 1683 ZP_01452386.1 1884 Mariprofundus 50467 . . . 52602 ferrooxydans PV-1 94500866 NZ_AAQH01000011.1: 1684 ZP_01307392.1 1885 Bermanella 33324 . . . 35456 marisrubri

In other embodiments, a polynucleotide, gene and/or polypeptide encoding phosphotransacetylase can have at least about 70% to about 75%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to any one of the sequences of Tables 10 or 12a or 12b, wherein the polynucleotide, gene and/or polypeptide encodes a polypeptide having phosphotransacetylase activity.

In embodiments, a polynucleotide, gene and/or polypeptide encoding phosphotransacetylase corresponds to the Enzyme Commission Number EC 2.3.1.8.

In other embodiments, the phosphotransacetylase polynucleotide, gene and/or polypeptide sequences described herein or those recited in the art can be used to identify phosphotransacetylase sequences or phosphotransacetylase homologs in other cells, as described above for PDC.

Methods for gene expression in recombinant host cells, including, but not limited to, yeast cells are known in the art (see, for example, Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). In embodiments, the coding region for the phosphoketolase and/or phosphotransacetylase genes to be expressed can be codon optimized for the target host cell, as well known to one skilled in the art. Expression of genes in recombinant host cells, including but not limited to yeast cells, can require a promoter operably linked to a coding region of interest, and a transcriptional terminator. A number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, and GPM1; and the following inducible promoters suitable for use in yeast: GAL1, GAL10 and CUP1. Other yeast promoters include hybrid promoters UAS(PGK1)-FBA1p (SEQ ID NO: 1893), UAS(PGK1)-ENO2p (SEQ ID NO: 1894), UAS(FBA1)-PDC1p (SEQ ID NO: 1895), UAS(PGK1)-PDC1p (SEQ ID NO: 1896), and UAS(PGK)-OLE1p (SEQ ID NO: 1897). Suitable transcriptional terminators that can be used in a chimeric gene construct for expression include, but are not limited to, FBA1t, TDH3t, GPM1t, ERG10t, GAL1t, CYC1t, and ADH1t.

Recombinant polynucleotides are typically cloned for expression using the coding sequence as part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a termination control region. The coding region may be from the host cell for transformation and combined with regulatory sequences that are not native to the natural gene encoding phosphoketolase and/or phosphotransacetylase. Alternatively, the coding region may be from another host cell.

Vectors useful for the transformation of a variety of host cells are common and described in the literature. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors can comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.

In embodiments, suitable promoters, transcriptional terminators, and phosphoketolase and/or phosphotransacetylase coding regions can be cloned into E. coli-yeast shuttle vectors, and transformed into yeast cells. Such vectors allow strain propagation in both E. coli and yeast strains, and can contain a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. Typically used plasmids in yeast include, but are not limited to, shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2-micron origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are HIS3 (vector pRS423), TRP1 (vector pRS424), LEU2 (vector pRS425) and URA3 (vector pRS426).

In embodiments, construction of expression vectors with a chimeric gene encoding the described phosphoketolases and/or phosphotransacetylases can be performed by the gap repair recombination method in yeast. In embodiments, a yeast vector DNA is digested (e.g., in its multiple cloning site) to create a “gap” in its sequence. A number of insert DNAs of interest are generated that contain an approximately 21 bp sequence at both the 5′ and the 3′ ends that sequentially overlap with each other, and with the 5′ and 3′ terminus of the vector DNA. For example, to construct a yeast expression vector for “Gene X,” a yeast promoter and a yeast terminator are selected for the expression cassette. The promoter and terminator are amplified from the yeast genomic DNA, and Gene X is either PCR amplified from its source organism or obtained from a cloning vector comprising Gene X sequence. There is at least a 21 bp overlapping sequence between the 5′ end of the linearized vector and the promoter sequence, between the promoter and Gene X, between Gene X and the terminator sequence, and between the terminator and the 3′ end of the linearized vector. The “gapped” vector and the insert DNAs are then co-transformed into a yeast strain and plated on the medium containing the appropriate compound mixtures that allow complementation of the nutritional selection markers on the plasmids. The presence of correct insert combinations can be confirmed by PCR mapping using plasmid DNA prepared from the selected cells. The plasmid DNA isolated from yeast (usually low in concentration) can then be transformed into an E. coli strain, e.g. TOP10, followed by mini preps and restriction mapping to further verify the plasmid construct. Finally the construct can be verified by sequence analysis.

Like the gap repair technique, integration into the yeast genome also takes advantage of the homologous recombination system in yeast. In embodiments, a cassette containing a coding region plus control elements (promoter and terminator) and auxotrophic marker is PCR-amplified with a high-fidelity DNA polymerase using primers that hybridize to the cassette and contain 40-70 base pairs of sequence homology to the regions 5′ and 3′ of the genomic area where insertion is desired. The PCR product is then transformed into yeast and plated on medium containing the appropriate compound mixtures that allow selection for the integrated auxotrophic marker. For example, to integrate “Gene X” into chromosomal location “Y”, the promoter-coding region X-terminator construct is PCR amplified from a plasmid DNA construct and joined to an autotrophic marker (such as URA3) by either SOE PCR or by common restriction digests and cloning. The full cassette, containing the promoter-coding regionX-terminator-URA3 region, is PCR amplified with primer sequences that contain 40-70 bp of homology to the regions 5′ and 3′ of location “Y” on the yeast chromosome. The PCR product is transformed into yeast and selected on growth media lacking uracil. Transformants can be verified either by colony PCR or by direct sequencing of chromosomal DNA.

The presence of phosphoketolase and phosphotransacetylase activity in the recombinant host cells disclosed herein can be confirmed using routine methods known in the art. In a non-limiting example, and as described in the Examples herein, transformants can be screened by PCR using primers for the phosphoketolase and phosphotransacetylase genes. In embodiments, and as described in the Examples herein, transformants can be screened by PCR with primers N1039 and N1040 (SEQ ID NOs: 639 and 640) to confirm integration of the xpk1 gene, and primers N1041 and N1042 (SEQ ID NOs: 641 and 642) can be used to confirm integration of the eutD gene. In another non-limiting example, and as described in the Examples herein, transformants can be screened for integration of phosphoketolase constructs and/or phosphotransacetylase constructs at the Δpdc1::ilvD(Sm) locus by the loss of ilvD(Sm) in the host cells.

In another non-limiting example, and as described in the examples herein, phosphoketolase activity can be assayed by expressing phosphoketolase identifiable by the methods disclosed herein in a recombinant host cell disclosed herein that lacks endogenous phosphoketolase activity. If phosphoketolase activity is present, such cells exhibit a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for growth in culture.

In another non-limiting example, and as described in the examples herein, phosphoketolase and phosphotransacetylase activity can be assayed by expressing phosphoketolase and phosphotransacetylase activity identifiable by the methods disclosed herein in a recombinant host cell disclosed herein that lacks endogenous phosphoketolase and phosphotransacetylase activity. If phosphoketolase activity and phosphoketolase activity are present, such cells exhibit a reduced or eliminated requirement for exogenous two-carbon substrate supplementation for growth in culture.

In another non-limiting example, phosphoketolase and/or phosphotransacetylase activity can be confirmed by more indirect methods, such as by assaying for a downstream product in a pathway requiring phosphoketolase activity. For example, a polypeptide having phosphoketolase activity can catalyze the conversion of xylulose-5-phosphate into glyceraldehyde-3-phosphate and acetyl-phosphate and/or the conversion of fructose-6-phosphate into erythrose-4-phosphate and acetyl-phosphate. Also, a polypeptide having phosphotransacetylase activity can catalyze the conversion of acetyl-phosphate into acetyl-CoA.

Suitable Pathway Carbon Substrates and Exogenous Two-Carbon Substrate Supplementation

PDC-KO cells fail to grow in glucose-containing media (e.g., 2% glucose), but PDC-KO cells carrying a functional butanediol biosynthetic pathway have been shown to grow on glucose supplemented with exogenous two-carbon substrates such as ethanol (see for example, US Patent Application Publication No. 20090305363, herein incorporated by reference). In embodiments, the host cells disclosed herein can be grown in fermentation media which contains a suitable pathway carbon substrate and two-substrate supplement, including combinations of suitable pathway carbon substrates with C2-substrate supplement. Non-limiting examples of suitable pathway carbon substrates include, but are not limited to, monosaccharides such as fructose, oligosaccharides such as lactose maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt, including any combinations thereof. In other embodiments, the suitable pathway carbon substrates can include lactate, glycerol, or combinations thereof.

In embodiments, a suitable carbon substrate can be a one-carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated, or combinations thereof. In other embodiments related to methylotrophic organisms, the carbon substrate can be carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. In a non-limiting example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). In another non-limiting example, various species of Candida can metabolize alanine (Sulter et al., Arch. Microbiol. 153:485-489 (1990)). Hence it is contemplated that the source of carbon utilized in the present invention can encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

In other embodiments, the suitable pathway carbon substrate can be glucose, fructose, and sucrose, or mixtures of these with five-carbon (C5) sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. In embodiments, sucrose can be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. In other embodiment, glucose and dextrose can derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In embodiments, the pathway carbon substrates can be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Application Publication No. US 20070031918 A1, which is herein incorporated by reference.

As used herein, “biomass” refers to any cellulosic or lignocellulosic material and includes, but is not limited to, materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. In embodiments, biomass can also comprise additional components, such as protein and/or lipid. In other embodiments, biomass can be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass can comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Other non-limiting examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.

The recombinant host cells described herein can be cultured using standard laboratory techniques known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In embodiments related to media supplemented with exogenous two-carbon substrates, and as described in the Examples, recombinant host cells can be grown in synthetic complete medium supplemented with one or more exogenous two-carbon substrates as described herein at a concentration of about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 1.5% or about 2% (v/v) of the media. In embodiments, the recombinant host cells can be grown in synthetic complete culture without uracil or histidine, supplemented with 0.5% (v/v) ethanol. In embodiments related to growth in media that is not supplemented with exogenous two-carbon substrates, the recombinant host cells described herein can be first grown in culture medium comprising an exogenous two-carbon substrate and then diluted (e.g., starting OD=0.1, 20 ml medium in a 125 ml vented flask) into media that is not supplemented with exogenous two-carbon substrate.

The growth of the recombinant host cells described herein can be measured by methods known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In a non-limiting example, the growth of the recombinant host cells described herein can be determined by measuring the optical density (OD) of cell cultures over time. For example, the OD at 600 nm for a yeast culture is proportional to yeast cell number. In another non-limiting example, the growth of the recombinant host cells described herein can be determined by counting viable cells in a sample of the culture over time.

Applicants have provided cells that have a reduced or eliminated requirement for two-carbon substrate supplementation for growth. In embodiments, such cells comprise (i) a deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA that results in a requirement for exogenous two-carbon substrate supplementation for optimal growth; (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. In embodiments, such cells comprise (i) a modification in an endogenous polypeptide having PDC activity which results in reduced or eliminated PDC activity; (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and optionally (iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity. As such, Applicants have also provided methods of improving the growth of a recombinant host cell comprising at least one modification in an endogenous polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA that results in a requirement for exogenous two-carbon substrate supplementation for optimal growth comprising transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. Applicants have also provided methods of improving the growth of a recombinant host cell comprising at least one modification in an endogenous polypeptide having pyruvate decarboxylase activity (e.g., having at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having PDC activity that results in reduced or eliminated PDC activity) comprising transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the method further comprises transforming a recombinant host cell described herein with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Applicants have also provided methods of reducing or eliminating the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one modification in an endogenous activity that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA that results in a requirement for exogenous two-carbon substrate supplementation for optimal growth comprising transforming the host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity comprising transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the method further comprises transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

Applicants have also provided methods of reducing the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one modification in an endogenous polypeptide having PDC activity (e.g., having at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity) comprising transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the method further comprises transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

In addition, Applicants have provided methods of eliminating the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one modification in an endogenous polypeptide having PDC activity (e.g., having at least one deletion, mutation or substitution in an endogenous gene encoding a polypeptide having PDC activity that results in reduced or eliminated PDC activity) comprising transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the method further comprises transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

In embodiments, a reduced requirement for exogenous two-carbon substrate supplementation can be a growth rate of the recombinant host cells described herein in media that is not supplemented with an exogenous two-carbon substrate that is the same or substantially equivalent to the growth rate of a recombinant host cell comprising a modification in an endogenous activity that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA grown in media that is supplemented with an exogenous two-carbon substrate. In embodiments, such a growth rate can be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the growth rate of a recombinant host cell comprising a modification in an endogenous activity that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA grown in media that is supplemented with an exogenous two-carbon substrate.

In embodiments, a reduced requirement for exogenous two-carbon substrate supplementation can be a growth rate of the recombinant host cells described herein in media that is not supplemented with an exogenous two-carbon substrate that is the same or substantially equivalent to the growth rate of a recombinant host cell comprising a modification in an endogenous PDC activity grown in media that is supplemented with an exogenous two-carbon substrate. In embodiments, such a growth rate can be at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the growth rate of a recombinant host cell comprising a modification in an endogenous PDC activity grown in media that is supplemented with an exogenous two-carbon substrate.

In other embodiments, the recombinant host cells described herein have a growth rate in media that is not supplemented with an exogenous two-carbon substrate that is greater than the growth rate of a recombinant host cell comprising a modification in an endogenous activity that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA in media that is not supplemented with an exogenous two-carbon substrate.

In other embodiments, the recombinant host cells described herein have a growth rate in media that is not supplemented with an exogenous two-carbon substrate that is greater than the growth rate of a recombinant host cell comprising a modification in an endogenous PDC activity in media that is not supplemented with an exogenous two-carbon substrate.

In other embodiments, the recombinant host cells described herein can have an increased glucose consumption compared to a recombinant host cell comprising a modification in an endogenous polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA.

In other embodiments, the recombinant host cells described herein can have an increased glucose consumption compared to a recombinant host cell comprising a modification in an endogenous polypeptide having PDC activity (e.g., at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having PDC activity that reduces or eliminates PDC activity).

Glucose consumption of the recombinant host cells described herein can be measured by methods known in the art (see, e.g., Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). In a non-limiting example, glucose consumption can be measured by quantitating the amount of glucose in culture media by HPLC or with a YSI Biochemistry Analyzer (YSI, Inc., Yellow Springs, Ohio).

In other embodiments, methods of producing a recombinant host cell are provided comprising transforming a recombinant host cell comprising a modification in an endogenous polynucleotide, gene or polypeptide encoding pyruvate decarboxylase (e.g., at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity) with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity. In other embodiments, the method further comprises transforming the recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

In other embodiments, methods for the conversion of xylulose 5-phosphate or fructose 6-phosphate into acetyl-phosphate are provided comprising (i) providing a recombinant host cell as described herein, or combinations thereof; and (ii) growing the recombinant host cell under conditions wherein xylulose 5-phosphate or fructose-6-phosphate is converted into acetyl-phosphate. In other embodiments, methods for the conversion of xylulose 5-phosphate or fructose-6-phosphate into acetyl-CoA are provided comprising (i) providing a recombinant host cell as described herein, or combinations thereof; and (ii) growing the recombinant host cell under conditions where xylulose 5-phosphate or fructose-6-phosphate is converted into acetyl-CoA.

In other embodiments, methods for the conversion of acetyl-phosphate to acetyl-CoA are provided comprising (i) providing a recombinant host cell as described herein, or combinations thereof; and (ii) growing the recombinant host cell under conditions where acetyl-phosphate is converted into acetyl-CoA. In other embodiments, methods for increasing the specific activity of a heterologous polypeptide having phosphoketolase activity in a recombinant host cell are provided comprising (i) providing a recombinant host cell as described herein, or combinations thereof; and (ii) growing the recombinant host cell under conditions wherein the heterologous polypeptide having phosphoketolase activity is expressed in functional form having a specific activity greater than the same recombinant host cell lacking the heterologous polypeptide having phosphoketolase activity.

In other embodiments, methods for increasing the specific activity of a heterologous polypeptide having phosphotransacetylase activity in a recombinant host cell are provided comprising (i) providing a recombinant host cell described herein, or combinations thereof; and (ii) growing the recombinant host cell under conditions whereby the heterologous polypeptide having phosphotransacetylase activity is expressed in functional form having a specific activity greater than the same recombinant host cell lacking a heterologous polypeptide having phosphotransacetylase activity.

In still other embodiments, methods for increasing the activity of the phosphoketolase pathway in a recombinant host cell are provided comprising (i) providing a recombinant host cell as described herein, or combinations thereof; and (ii) growing the host cell under conditions whereby the activity of the phosphoketolase pathway in the host cell is increased.

Threonine aldolase (E.C. number 4.1.2.5) catalyzes cleavage of threonine to produce glycine and acetaldehyde. Plasmid-based overexpression of a gene encoding this enzyme in S. cerevisiae PDC-KO strains was shown to eliminate the requirement for exogenous C2 supplementation (van Maris et al, Appl Environ Microbiol. 2003 April; 69(4):2094-9). In embodiments, recombinant host cells comprise (i) a deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA that results in a requirement for exogenous two-carbon substrate supplementation for optimal growth; and (ii) a heterologous polynucleotide encoding a polypeptide having threonine aldolase activity.

Engineered Biosynthetic Pathways Using Pyruvate

In embodiments, the recombinant host cells described herein can be engineered to have a biosynthetic pathway for production of a product from pyruvate. A product from such a pyruvate-utilizing biosynthetic pathway includes, but is not limited to, 2,3-butanediol, isobutanol, 2-butanol, 2-butanone, valine, leucine, alanine, lactic acid, malic acid, fumaric acid, succinic acid and isoamyl alcohol. The features of any pyruvate-utilizing biosynthetic pathway may be engineered in the recombinant host cells described herein in any order. Any product made using a biosynthetic pathway that has pyruvate as the initial substrate can be produced with greater effectiveness in a recombinant host cell disclosed herein having a modification in an endogenous polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA (such as pyruvate decarboxylase, pyruvate formate lyase, pyruvate dehydrogenase, pyruvate oxidase, or pyruvate:ferredoxin oxidoreductase) and having heterologous phosphoketolase and/or phosphotransacetylase activity, compared to a recombinant host cell having a modification in an endogenous polypeptide that converts pyruvate to acetaldehyde, acetyl-phosphate or acetyl-CoA (such as pyruvate decarboxylase, pyruvate formate lyase, pyruvate dehydrogenase, pyruvate oxidase, or pyruvate:ferredoxin oxidoreductase). Any product made using a biosynthetic pathway that has pyruvate as the initial substrate can be produced with greater effectiveness in a recombinant host cell disclosed herein having a modification in an endogenous polypeptide having PDC activity that reduces or eliminates PDC activity and having heterologous phosphoketolase and/or phosphotransacetylase activity, compared to a recombinant host cell having a modification in an endogenous polypeptide having PDC activity that reduces or eliminates PDC activity.

The biosynthetic pathway of the recombinant host cells described herein can be any pathway that utilizes pyruvate and produces a desired product. The pathway genes may include endogenous genes and/or heterologous genes. Typically at least one gene in the biosynthetic pathway is a heterologous gene. Suitable biosynthetic pathways for production of butanol are known in the art, and certain suitable pathways are described herein. In some embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises more than one gene that is heterologous to the host cell. In some embodiments, the butanol biosynthetic pathway comprises heterologous genes encoding polypeptides corresponding to every step of a biosynthetic pathway.

Genes and polypeptides that can be used for substrate to product conversions described herein as well as methods of identifying such genes and polypeptides, are described herein and/or in the art, for example, for isobutanol, in the Examples and in U.S. Pat. No. 7,851,188. Ketol-acid reductoisomerase (KARI) enzymes are described in U.S. Patent Appl. Pub. Nos. 20080261230 A1, 20090163376 A1, 20100197519 A1, and PCT Appl. Pub. No. WO/2011/041415. Examples of KARIs disclosed therein are those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, and Pseudomonas fluorescens PF5 mutants. KARIs include Anaerostipes caccae KARI variants “K9G9” and “K9D3” (SEQ ID NOs: 1911 and 1910, respectively). US Appl. Pub. No. 20100081154 A1, and U.S. Pat. No. 7,851,188 describe dihydroxyacid dehydratases (DHADs), including a DHAD from Streptococcus mutans. U.S. Patent Appl. Publ. No. 20090269823 A1 describes SadB, an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol dehydrogenases also include horse liver ADH and Beijerinckia indica ADH (protein SEQ ID NO: 1923).

An example of a biosynthetic pathway for producing 2,3-butanediol can be engineered in the recombinant host cells described herein, as described in U.S. Patent Application No. 20090305363, which is herein incorporated by reference. The 2,3-butanediol pathway is a portion of the 2-butanol biosynthetic pathway that is disclosed in U.S. Patent Application Publication No. US 20070292927 A1, which is herein incorporated by reference. Such pathway steps include, but are not limited to, conversion of pyruvate to acetolactate by acetolactate synthase, conversion of acetolactate to acetoin by acetolactate decarboxylase, and conversion of acetoin to 2,3-butanediol by butanediol dehydrogenase. The skilled person will appreciate that polypeptides having the activity of such pathway steps can be isolated from a variety of sources can be used in the recombinant host cells described herein.

In addition, examples of biosynthetic pathways for production of 2-butanone or 2-butanol that can be engineered in the recombinant host cells described herein are disclosed in U.S. Patent Application Publication Nos. US 20070292927 A1 and US 20070259410 A1, which are herein incorporated by reference. The pathway in U.S. Patent Application Publication No. US 20070292927 A1 is the same as described for butanediol production with the addition of the following steps:

-   -   2,3-butanediol to 2-butanone as catalyzed for example by diol         dehydratase or glycerol dehydratase; and     -   2-butanone to 2-butanol as catalyzed for example by butanol         dehydrogenase.

Described in U.S. Patent Application Publication No. US 20090155870 A1, which is herein incorporated by reference, is the construction of chimeric genes and genetic engineering of yeast for 2-butanol production using the U.S. Patent Application Publication No. US 20070292927 A1 disclosed biosynthetic pathway. Further description for gene construction and expression related to these pathways can be found, for example, in International Publication No. WO 2009046370 (e.g., butanediol dehydratases); and U.S. Patent Application Publication No. US 20090269823 A1 (e.g., butanol dehydrogenase) and U.S. Patent Application Publication No. US 20070259410 A1 which are herein incorporated by reference. The skilled person will appreciate that polypeptides having the activity of such pathway steps can be isolated from a variety of sources can be used in the recombinant host cells described herein.

Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Pat. No. 7,851,188 and PCT Publication WO 2007050671, incorporated herein by reference. One isobutanol biosynthetic pathway comprises the following substrate to product conversions:

-   -   pyruvate to acetolactate, which may be catalyzed, for example,         by acetolactate synthase;     -   acetolactate to 2,3-dihydroxyisovalerate, which may be         catalyzed, for example, by acetohydroxy acid reductoisomerase;     -   2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be         catalyzed, for example, by acetohydroxy acid dehydratase;     -   α-ketoisovalerate to isobutyraldehyde, which may be catalyzed,         for example, by a branched-chain keto acid decarboxylase; and     -   isobutyraldehyde to isobutanol, which may be catalyzed, for         example, by a branched-chain alcohol dehydrogenase.

In some embodiments, the isobutanol biosynthetic pathway comprises at least one gene, at least two genes, at least three genes, or at least four genes that is/are heterologous to the yeast cell. In embodiments, each substrate to product conversion of an isobutanol biosynthetic pathway in a recombinant host cell is catalyzed by a heterologous polypeptide. In embodiments, the polypeptide catalyzing the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol are capable of utilizing NADH as a cofactor.

An example of a biosynthetic pathway for production of valine that can be engineered in the recombinant host cells described herein includes the steps of acetolactate conversion to 2,3-dihydroxy-isovalerate by acetohydroxyacid reductoisomerase (ILV5), conversion of 2,3-dihydroxy-isovalerate to 2-keto-isovalerate by dihydroxy-acid dehydratase (ILV3), and conversion of 2-keto-isovalerate to valine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). Biosynthesis of leucine includes the same steps to 2-keto-isovalerate, followed by conversion of 2-keto-isovalerate to alpha-isopropylmalate by alpha-isopropylmalate synthase (LEU9, LEU4), conversion of alpha-isopropylmalate to beta-isopropylmalate by isopropylmalate isomerase (LEU1), conversion of beta-isopropylmalate to alpha-ketoisocaproate by beta-IPM dehydrogenase (LEU2), and finally conversion of alpha-ketoisocaproate to leucine by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1). It is desired for production of valine or leucine to overexpress at least one of the enzymes in these described pathways.

An example of a biosynthetic pathway for production of isoamyl alcohol that can be engineered in the recombinant host cells described herein includes the steps of leucine conversion to alpha-ketoisocaproate by branched-chain amino acid transaminase (BAT2) and branched-chain amino acid aminotransferase (BAT1), conversion of alpha-ketoisocaproate to 3-methylbutanal by ketoisocaproate decarboxylase (THI3) or decarboxylase ARO10, and finally conversion of 3-methylbutanal to isoamyl alcohol by an alcohol dehydrogenase such as ADH1 or SFA1. Production of isoamyl alcohol benefits from increased production of leucine or the alpha-ketoisocaproate intermediate by overexpression of one or more enzymes in biosynthetic pathways for these chemicals. In addition, one or both enzymes for the final two steps can be overexpressed.

An example of a biosynthetic pathway for production of lactic acid that can be engineered in the recombinant host cells described herein includes pyruvate conversion to lactic acid by lactate dehydrogenase. Engineering yeast for lactic acid production using lactate dehydrogenase, known as EC 1.1.1.27, is well known in the art such as in Ishida et al. (Appl. Environ. Microbiol. 71:1964-70 (2005)).

An example of a biosynthetic pathway for production of alanine that can be engineered in the recombinant host cells described herein includes pyruvate conversion to alanine by aminotransferase.

An example of a biosynthetic pathway for production of malate that can be engineered in the recombinant host cells described herein includes pyruvate conversion to oxaloacetate by pyruvate carboxylase, and conversion of oxaloacetate to malate by malate dehydrogenase as described in Zelle et al. (Applied and Environmental Microbiology 74:2766-77 (2008)). In addition, a malate transporter can be expressed.

An example of a biosynthetic pathway for production of fumarate that can be engineered in the recombinant host cells described herein includes pyruvate conversion to oxaloacetate by pyruvate carboxylase, and conversion of oxaloacetate to malate by malate dehydrogenase as described in Zelle et al. (Applied and Environmental Microbiology 74:2766-77 (2008)). In addition, a fumarase and a fumarate transporter can be expressed. Favorable production conditions and engineering of fungi for fumarate production is well known in the art, described e.g. by Goldberg et al. (Journal of Chemical Technology and Biotechnology 81:1601-1611 (2006)).

An example of a biosynthetic pathway for production of succinate that can be engineered in the recombinant host cells described herein includes pyruvate conversion to oxaloacetate by pyruvate carboxylase, and conversion of oxaloacetate to malate by malate dehydrogenase as described in Zelle et al. (Applied and Environmental Microbiology 74:2766-77 (2008)). In addition, a fumarase, a succinate dehydrogenase and a succinate transporter can be expressed.

The skilled person will appreciate that polypeptides having activities of the above-mentioned biosynthetic pathways can be isolated from a variety of sources can be used in the recombinant host cells described herein.

It will be appreciated that host cells comprising a butanol biosynthetic pathway such as an isobutanol biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Appl. Pub. No. 20090305363 (incorporated by reference) discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. Modifications to reduce glycerol-3-phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Patent Appl. Pub. No. 20090305363 (incorporated herein by reference), modifications to a host cell that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in U.S. Patent Appl. Pub. No. 20100120105 (incorporated herein by reference). Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway. Other modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In embodiments, the polypeptide having acetolactate reductase activity is YMR226C (SEQ ID NO: 1912) of Saccharomyces cerevisiae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 (SEQ ID NO: 1909) from Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Appl. Publication No. 20110124060, incorporated herein by reference.

Recombinant host cells may further comprise (a) at least one heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity; and (b)(i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe-S cluster biosynthesis; and/or (ii) at least one heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster biosynthesis. In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is encoded by AFT1 (nucleic acid SEQ ID NO: 1913, amino acid SEQ ID NO: 1914), AFT2 (SEQ ID NOs: 1915 and 1916), FRA2 (SEQ ID NOs: 1917 and 1918), GRX3 (SEQ ID NOs: 1919 and 1920), or CCC1 (SEQ ID NOs: 1921 and 1922). In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT1 C293F.

Fermentation Media

The recombinant host cells disclosed herein can be grown in fermentation media for production of a product utilizing pyruvate. For maximal production of some products, such as 2,3-butanediol, isobutanol, 2-butanone, or 2-butanol, the recombinant host cells disclosed herein used as production hosts preferably have enhanced tolerance to the produced chemical, and have a high rate of carbohydrate utilization. These characteristics can be conferred by mutagenesis and selection, genetic engineering, or can be natural.

Fermentation media for production of the products disclosed herein may contain glucose. Additional carbon substrates for product production pathways can include but are not limited to those described above. It is contemplated that the source of carbon utilized can encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

In addition to an appropriate carbon source, fermentation media can contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for production of the desired product.

Culture Conditions

Typically cells are grown at a temperature in the range of about 20° C. to about 37° C. in an appropriate medium. Suitable growth media for the recombinant host cells described herein are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most Saccharomyces cerevisiae strains. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.

Suitable pH ranges for the fermentation are between pH 3.0 to pH 7.5, where pH 4.5 to pH 6.5 is preferred as the initial condition.

Fermentations can be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

The amount of product in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).

Industrial Batch and Continuous Fermentations

A batch method of fermentation can be used with the recombinant host cells described herein. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.

A Fed-Batch system can also be used with the recombinant host cells described herein. A Fed-Batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO₂. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.

Although a batch mode can be performed, it is also contemplated that continuous fermentation methods could also be performed with the recombinant host cells described herein. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the present invention can be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells can be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.

Product Isolation from Fermentation Medium

Products can be isolated from the fermentation medium by methods known to one skilled in the art. For example, bioproduced isobutanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the isobutanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, pervaporation or vacuum flash fermentation (see e.g., U.S. Pub. No. 20090171129 A1, and International Pub. No. WO2010/151832 A1, both incorporated herein by reference in their entirety).

Because butanol forms a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the isobutanol. In this method, the isobutanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the isobutanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The isobutanol-rich decanted organic phase may be further purified by distillation in a second distillation column.

The butanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the isobutanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The isobutanol-containing organic phase is then distilled to separate the butanol from the solvent.

Distillation in combination with adsorption can also be used to isolate isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy Laboratory, June 2002).

Additionally, distillation in combination with pervaporation may be used to isolate and purify the isobutanol from the fermentation medium. In this method, the fermentation broth containing the isobutanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo et al., J. Membr. Sci. 245, 199-210 (2004)).

In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields. One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction. In general, with regard to butanol fermentation, for example, the fermentation medium, which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the fermentation medium form a biphasic mixture. The butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.

Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 20090305370, the disclosure of which is hereby incorporated in its entirety. U.S. Patent Appl. Pub. No. 20090305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, and mixtures thereof. The extractant(s) for ISPR can be non-alcohol extractants. The ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.

In some embodiments, the alcohol can be esterified by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst (e.g. enzyme such as a lipase) capable of esterifying the alcohol with the organic acid. In such embodiments, the organic acid can serve as an ISPR extractant into which the alcohol esters partition. The organic acid can be supplied to the fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol. The catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock. When the catalyst is supplied to the fermentation vessel, alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel. Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant. The extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration. In addition, unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.

In situ product removal can be carried out in a batch mode or a continuous mode. In a continuous mode of in situ product removal, product is continually removed from the reactor. In a batchwise mode of in situ product removal, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. For in situ product removal, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some embodiments of the present invention, the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel. The ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved. In some embodiments, the ester-containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.

EXAMPLES

The meaning of abbreviations used is as follows: “min” means minute(s), “h” means hour(s), “sec” means second(s), “μl” means microliter(s), “ml” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “rpm” means revolutions per minute, “w/v” means weight/volume, “v/v” means volume/volume, “OD” means optical density, “bp” means base pair(s), and “PCR” means polymerase chain reaction.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Phusion® HF Master Mix (NEB Cat. No. F-531) and HotStarTaq® Master Mix (Qiagen Cat. No. 203443) were used for PCR in gene cloning and clone screening, respectively.

Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Wash., D C., 1994, or by Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, Mass., 1989. All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), Life Technologies (Rockville, Md.), or Sigma Chemical Company (St. Louis, Mo.), unless otherwise specified.

HPLC

Analysis for fermentation by-product composition is well known to those skilled in the art. For example, one high performance liquid chromatography (HPLC) method utilizes a Shodex SH-1011 column with a Shodex SH-G guard column (both available from Waters Corporation, Milford, Mass.), with refractive index (RI) detection. Chromatographic separation is achieved using 0.01 M H₂SO₄ as the mobile phase with a flow rate of 0.5 mL/min and a column temperature of 50° C. Isobutanol retention time is 47.6 minutes. For butanediol, meso-butanediol eluted at 26.0 min and 2R,3R-butanediol eluted at 27.7 min.

Example 1 Construction of Phosphoketolase/Phosphotransacetylase Expression Cassette

The xpk1 and eutD genes (GenBank GI numbers 28379168 (SEQ ID NO: 172) and 28377658 (SEQ ID NO: 1111), respectively) were obtained from Lactobacillus plantarum (ATCC No. BAA-793) via polymerase chain reaction (PCR) using primers N1039 and N1040 (for xpk1) and N1041 and N1042 (for eutD). The primer sequences of N1039, N1040, N1041 and N1042 correspond to SEQ ID Nos. 639-642, respectively.

The xpk1 and eutD genes were fused to a DNA fragment containing opposing yeast terminator sequences (CYC and ADH terminators, obtained from PacI digestion of pRS423::CUP1-alsS+FBA-budA, described in U.S. Patent Application Publication No. 20090155870, herein incorporated by reference) by overlap PCR method (Yu et al., Fungal Genet. Biol. 41: 973-981; 2003). The resulting PCR product was cloned into an E. coli-yeast shuttle vector using gap repair methodology (Ma et al., Genetics 58:201-216; 1981). The shuttle vector was based on pRS426 (ATCC No. 77107) and contained both GPD (also known as TDH3) and ADH1 promoters. The resulting vector contained xpk1 under control of the GPD promoter and eutD under control of the ADH1 promoter in opposing orientation. The sequence of the resulting vector (pRS426::GPD-xpk1+ADH1-eutD) is provided as SEQ ID No: 643 (see FIG. 5 for a map of this vector).

Example 2 Construction of Phosphoketolase/Phosphotransacetylase Integration Vector

An expression cassette of the pRS426::GPD-xpk1+ADH1-eutD vector (GPD-xpk1+ADH1-eutD) was prepared by digestion with EcoRI and SacI restriction enzymes. The resulting cassette was ligated into the yeast integration vector pUC19-URA3-MCS which was also prepared by digestion with EcoRI and SacI restriction enzymes.

Vector pUC19-URA3MCS is pUC19-based and contains the sequence of the URA3 gene from Saccharomyces cerevisiae situated within a multiple cloning site (MCS). pUC19 (American Type Culture Collection, Manassas, Va.; ATCC#37254) contains the pMB1 replicon and a gene coding for beta-lactamase for replication and selection in Escherichia coli. In addition to the coding sequence for URA3, the sequences from upstream and downstream of this gene are included for expression of the URA3 gene in yeast. The vector can be used for cloning purposes and can be used as a yeast integration vector.

The DNA encompassing the URA3 coding region along with 250 bp upstream and 150 bp downstream of the URA3 coding region from Saccharomyces cerevisiae CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) genomic DNA was amplified with primers oBP438 (SEQ ID NO: 644), containing BamHI, AscI, PmeI, and FseI restriction sites, and oBP439 (SEQ ID NO: 645), containing XbaI, PacI, and NotI restriction sites. Genomic DNA was prepared using a Gentra Puregene Yeast/Bact kit (Qiagen). The PCR product and pUC19 were ligated with T4 DNA ligase after digestion with BamHI and XbaI to create vector pUC19-URA3MCS. The vector was confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO:646) and oBP265 (SEQ ID NO:647).

The ligation reaction was transformed into E. coli Stb13 cells, according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., Cat. No. C7373). Transformants were screened by polymerase chain reaction (PCR) to detect the eutD gene using the primers N1041 and N1042 (SEQ ID NOs: 641 and 642, respectively). Positive clones for eutD gene expression detected by PCR were further confirmed for eutD gene incorporation by digestion of the vector with SacII restriction enzyme.

Two confirmed clones were selected and an integration targeting sequence was added to the clones as follows. PCR was used to amplify regions of the genome of S. cerevisiae strain BY4700 (ATCC No. 200866) both 5′ and 3′ of the PDC1 gene using the following primers: N1049 and N1050 (5′) and N1047 and N1048 (3′) (SEQ ID NOs: 648-651, respectively). Primer N1049 enables the 3′ end of the 161-bp PDC1 3′ sequence to be fused to the 5′ end of the 237 bp PDC1 5′ sequence via PCR. This pdc1 3′-5′-fusion fragment (368 bp in length) was cloned into the pCRII-Blunt TOPO vector according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., Cat. No. K2800).

Transformants were screened by PCR to detect the pdc1 3′-5′-fusion fragment using primers N1047 and N1050. The pdc1 3′-5′-fusion fragment was isolated from positive clones and released from the vector by digestion with EcoRI enzyme, and ligated into a pUC19-URA3::GPD-xpk1+ADH-eutD vector that had been linearized by digestion with EcoRI restriction enzyme to generate the “phosphoketolase pathway” vector. Additionally, the pdc1 3′-5′-fusion fragment was ligated with pUC19-URA3-MCS digested with EcoRI restriction enzyme to generate the control vector. Both ligation reactions were transformed into E. coli Stb13 cells according to the manufacturer's instructions (Invitrogen, Carlsbad, Calif., Cat. No. C7373). The resulting transformants were screened by PCR to detect the pdc1 3′-5′-fusion fragment using primers N1047 and N1050. Positive clones containing the pdc1 3′-5′-fusion fragment were identified and the vectors were digested with either NcoI restriction enzyme (control vector) or BsgI restriction enzyme (phosphoketolase pathway vector) to confirm cloning orientation. One control clone (=pUC19-URA3::pdc1) and one phosphoketolase pathway clone (=pUC19-URA3::pdc1::GPD-xpk1+ADH1-eutD; SEQ ID NO: 1898) were selected for integration.

Example 3 Construction of Pyruvate Decarboxylase Knockout (PDC-KO) Yeast Strain Containing Phosphoketolase and Phosphotransacetylase Genes

The control and phosphoketolase pathway vectors described in Example 2 were linearized with AflII restriction enzyme and transformed into strain BP913 (CEN.PK113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvD(Sm) Δpdc5::sadB) to form control and phosphoketolase pathway strains. Strain BP913 is further described in Example 10.

Transformed cells were plated on synthetic complete medium without uracil containing ethanol as the sole carbon source (1% vol/vol) and screened by PCR using primers N238 and oBP264 (SEQ ID Nos. 652 and 646, respectively to confirm integration at the pdc1 locus. Integration at the Δpdc1::ilvD(Sm) locus resulted in the loss of ilvD(Sm).

Example 4 Introduction of Phosphoketolase and Phosphotransacetylase Allows Growth of PDC-KO Cells without Exogenous Two-Carbon Substrate Supplementation

Pyruvate decarboxylase knockout (PDC-KO) yeast strains are unable to grow in media containing 2% glucose as the sole carbon source, but can grow in 2% glucose supplemented with ethanol as shown with a strain transformed with one or more plasmids encoding members of the butanediol pathway (described in U.S. Patent Application Publication No. 20090305363, herein incorporated by reference). To test whether the introduction of the phosphoketolase and phosphotransacetylase genes could support growth of PDC-KO cells, PDC-KO yeast were transformed with the phosphoketolase and phosphotransacetylase gene (as described in Example 3) and with the vector pRS423::CUP1-alsS+FBA-budA (described in U.S. Patent Application Publication No. 20090155870, herein incorporated by reference) encoding members of the butanediol pathway. After cultivation in media containing 2% glucose (synthetic complete minus his and ura) supplemented with 0.05% v/v ethanol, cultures were diluted into the same media lacking ethanol (starting OD=0.1, 20 ml medium in a 125 ml vented flask). For comparison, a control PDC-KO strain without introduction of the phosphoketolase and phosphotransacetylase genes was also diluted into medium supplemented with ethanol (0.05% vol/vol). The optical density at 600 nm was measured during growth (results shown in FIG. 2 and Table 13).

TABLE 13 Condition 0 h 16 h 22 h 41.3 h Strain OD OD OD OD xpk A 0.1 2.07 5.63 9.64 xpk B 0.1 2.44 5.93 9.78 xpkC 0.1 2.26 5.83 9.96 control A 0.1 0.47 0.5 0.822 control B 0.1 0.45 0.51 0.849 control C 0.1 0.5 0.52 0.879 cont A w/EtOH 0.1 2.01 5.49 11.44 cont B w/EtOH 0.1 2.16 5.7 11.5 cont C w/EtOH 0.1 2.12 5.76 11.76

The growth of PDC-KO yeast transformed with phosphoketolase and phosphotransacetylase in media that was not supplemented with ethanol (xpkA-xpkC, representing n=3 results) was indistinguishable from the growth of PDC-KO yeast strains grown in media containing 2% glucose that was supplemented with ethanol (cont A-cont C w/EtOH, representing n=3 results). The average growth rate of the phosphoketolase- and phosphotransacetylase-transformed strains under these conditions was 0.19 h⁻¹. A growth rate of 0.23 h⁻¹ for the phosphoketolase- and phosphotransacetylase-transformed strains was observed upon culturing under the same conditions with higher aeration (data not shown). PDC-KO yeast strains grown in media containing 2% glucose that was not supplemented with ethanol showed some growth in the first 16 hours, but then grew at a rate of only 0.01 h⁻¹ (control A-control C, representing n=3 results).

Example 5 Construction of Pyruvate Decarboxylase Knockout (PDC-KO) Yeast Strains Containing Either Phosphoketolase or Phosphotransacetylase Genes

The integration vector described above (pUC19-URA3::pdc1::GPD-xpk1+ADH1-eutD) was modified to eliminate either the xpk1 phosphoketolase gene or the eutD phosphotransacetylase gene. Specifically, to remove eutD, the integration vector was digested with the ClaI and SpeI restriction enzymes to remove a 0.6 kb region from the eutD coding sequence, forming the vector pUC19-URA3::pdc1::GPD-xpk1. To remove xpk1, the integration vector was digested with the SpeI and KpnI restriction enzymes to remove the 3.4 kb region from SpeI to KpnI, forming the vector pUC19-URA3::pdc1::ADH-eutD. The resulting vectors, were linearized with digestion with the AflII restriction enzyme and transformed into BP913/pRS423::CUP1-alsS+FBA-budA cells (described in Example 3). Transformed cells were screened by PCR to confirm integration at the pdc1 locus and cultured, as described above.

Example 6 Introduction of Phosphoketolase Allows Growth of PDC-KO Cells without Exogenous Two-Carbon Substrate Supplementation

To test whether the introduction of either the phosphoketolase or phosphotransacetylase genes could support the growth of PDC-KO cells, PDC-KO yeast were transformed with either the phosphoketolase or phosphotransacetylase genes (as described in Example 5) and with the vector pRS423::CUP1-alsS+FBA-budA encoding members of the butanediol pathway (as described in Example 4). After cultivation in media containing 2% glucose (synthetic complete minus his and ura) supplemented with 0.05% v/v ethanol, cultures were diluted into the same media lacking ethanol (starting OD=0.1, 20 ml medium in a 125 ml vented flask). For comparison, a PDC-KO strain without introduction of the phosphoketolase or phosphotransacetylase genes were grown under the same conditions. The optical density at 600 nm was measured during growth (results shown in FIG. 3 and Table 15).

TABLE 15 0 h 24 h Strain OD OD xpk1 + eutD 0.1 7.48 none (control) 0.1 0.575 eutD only 0.1 0.338 eutD only 0.1 0.28 xpk1 only 0.1 6.74 xpk1 only 0.1 7.26

The growth of PDC-KO yeast transformed with phosphoketolase in media that was not supplemented with exogenous carbon substrate (xpk1, FIG. 3) was indistinguishable from the growth of PDC-KO yeast transformed with phosphoketolase and phosphotransacetylase grown in media containing 2% glucose that was supplemented with ethanol (xpk1+eutD, FIG. 3). The growth of PDC-KO yeast transformed with phosphotransacetylase (eutD, FIG. 3) was not significantly improved compared to PDC-KO yeast strains in media that was not supplemented with exogenous two-carbon substrate (none, FIG. 3).

Example 7 Introduction of Phosphoketolase to PDC-KO Cells Increases Glucose Consumption and Butanediol Yield

To test the effects of introduction of phosphoketolase into PDC-KO cells on glucose consumption and butanediol yield, PDC-KO yeast were transformed with either (1) phosphoketolase and phosphotransacetylase (as described in Example 4) and the vector pRS423::CUP1-alsS+FBA-budA encoding members of the butanediol (BDO) pathway (as described in Example 4) (“Xpk” in Table 16 below); or with (2) the vector pRS423::CUP1-alsS+FBA-budA encoding members of the butanediol pathway (“Control” in Table 6 below).

After cultivation in medium containing 2% glucose (synthetic complete minus histidine and uracil) supplemented with 0.05% ethanol, Xpk and Control cultures were diluted into medium without ethanol (starting OD=0.1, 20 ml medium in a 125 ml vented flask). Glucose consumption and butanediol yield of Xpk and Control cultures were measured by HPLC analysis of culture media for amount of glucose and butanediol as shown in the Table below.

TABLE 16 Introduction of Phosphoketolase Increases Glucose Consumption and Butanediol Yield of PDC-KO Cells. Glucose consumed Butanediol Strains (mM) Molar Yield Xpk (n = 3) 73.9 ± 2.4 0.475 ± 0.001 Control (n = 3) 48.3 ± 0.6 0.359 ± 0.003

The glucose consumption of Xpk cells (n=3) was nearly twice the amount of glucose consumption of control strains (n=3). In addition, the butanediol molar yield of Xpk cells was increased compared to the butanediol molar yield of Control cells.

Example 8 Construction of an Additional Phosphoketolase Pathway Integration Vector

A phosphoketolase/phosphotransacetylase integration vector similar to the one described in Example 2 was constructed. In this case the xpk1 and eutD gene constructs were cloned so that they would be integrated immediately downstream of the Δpdc1::ilvD(Sm) locus of BP913. To do this, the intergenic region between ilvD(Sm) and TRX1 was amplified from BP913 genomic DNA using primers N1110 and N1111 (SEQ ID Nos. 653 and 654). This was cloned into pUC19-URA3-MCS at the PmeI site, as follows. The ilvD-TRX1 PCR product was phosphorylated with polynucleotide kinase (NEB Cat. No. M0201), the vector was prepared by digesting with PmeI and treating with calf intestinal phosphatase, the two fragments were ligated overnight and cloned into E. coli Stb13 cells. Clones were screened by PCR (using N1110 and N1111 primers) and then digested with BsgI to determine the orientation of the ilvD-TRX1 insertion. One clone from each orientation (pUC19-URA3::ilvD-TRX1 A and B) was carried over to the next step: addition of the xpk1/eutD expression cassette. The xpk1/eutD expression cassette from pRS426::GPD-xpk1+ADH1-eutD was obtained by digestion with BglII and EcoRV. The 5′ overhanging DNA was filled in using Klenow Fragment. pUC19-URA3::ilvD-TRX1 was linearized with AflII and the 5′ overhanging DNA was filled in using Klenow fragment. This vector was then ligated with the prepared xpk1/eutD cassette. Ligation reactions were transformed into E. coli Stb13 cells. Clones were screened using primers for eutD (N1041 and N1042) and then digested with BamHI to determine orientation of the xpk1/eutD cassette relative to the ilvD-TRX1 DNA sequence.

The URA3 marker gene was then replaced with a geneticin resistance marker as follows. A chimeric geneticin resistance gene was constructed that contained the Kluyveromyces lactis TEF1 promoter and terminator (TEF1p-kan-TEF1t gene, provided as SEQ ID No. 655). This gene was maintained in a pUC19 vector (cloned at the SmaI site). The kan gene was isolated from pUC19 by first digesting with KpnI, removal of 3′ overhanging DNA using Klenow Fragment (NEB, Cat. No. M212), digesting with HincII and then gel purifying the 1.8 kb gene fragment (Zymoclean™ Gel DNA Recovery Kit, Cat. No. D4001, Zymo Research, Orange, Calif.). The URA3 marker was removed from pUC19-URA3::ilvD::GPD-xpk1+ADH1-eutD::TRX1 (paragraph above) using NsiI and NaeI (the 3′ overhanging DNA from NsiI digestion was removed with Klenow fragment). The vector and kan gene were ligated overnight and transformed into E. coli Stb13 cells. Clones were screened by PCR using primers BK468 and either N1090 or N1113 (SEQ ID Nos. 656, 657, and 658, respectively)—positive PCR results indicate presence and orientation of kan gene. Clones in both orientations were digested with PmeI and transformed into BP913 with selection on yeast extract-peptone medium supplied with 1% (v/v) ethanol as carbon source and 200 μg/ml geneticin (G418). A single transformant was obtained, as confirmed by PCR (primers N886 and oBP264 for the 5′ end N1090 and oBP512 for the 3′ end, SEQ ID Nos. 659, 646, 657, and 660, respectively). FIG. 6 depicts the locus after integration of the plasmid.

Example 9 Construction of an Isobutanol-Producing Strain Carrying the Phosphoketolase Pathway

The strain described in Example 8 was transformed with 2 plasmids containing genes for an isobutanol pathway pYZ090 and pYZ067 (SEQ ID NOs: 1892 and 1891).

pYZ090 was constructed to contain a chimeric gene having the coding region of the alsS gene from Bacillus subtilis (nt position 457-2172) expressed from the yeast CUP1 promoter (nt 2-449) and followed by the CYC1 terminator (nt 2181-2430) for expression of ALS, and a chimeric gene having the coding region of the ilvC gene from Lactococcus lactis (nt 3634-4656) expressed from the yeast ILV5 promoter (2433-3626) and followed by the ILV5 terminator (nt 4682-5304) for expression of KARI.

pYZ067 was constructed to contain the following chimeric genes: 1) the coding region of the ilvD gene from S. mutans UA159 with a C-terminal Lumio tag (nt 2260-3972) expressed from the yeast FBA1 promoter (nt 1661-2250) followed by the FBA1 terminator (nt 40005-4317) for expression of dihydroxy acid dehydratase, 2) the coding region for horse liver ADH (nt 4680-5807) expressed from the yeast GPM1 promoter (nt 5819-6575) followed by the ADH1 terminator (nt 4356-4671) for expression of alcohol dehydrogenase, and 3) the coding region of the kivD gene from Lactococcus lactis (nt 7175-8821) expressed from the yeast TDH3 promoter (nt 8830-9493) followed by the TDH3 terminator (nt 6582-7161) for expression of ketoisovalerate decarboxylase.

Transformants were obtained on synthetic complete medium lacking uracil and histidine with 1% (v/v) ethanol as carbon source and 100 μg/ml geneticin. Control strains (BP913) were also transformed with the same plasmids and plated without geneticin. A number of transformants were then patched to the same medium containing 2% glucose as carbon source and supplemented with 0.05% (v/v) ethanol. After 36 hours, patches were used to inoculate liquid medium (same composition as the plates). After 48 hours, ODs for both phosphoketolase pathway and control strains were similar (ca. 4-5 OD) and all were subcultured into medium lacking ethanol (i.e. no exogenous two-carbon substrate source). The phosphoketolase cultures grew without ethanol supplementation, similar to ethanol supplemented control strains. Results are shown in FIG. 7A (and Table 17A). These were subcultured again to confirm growth rates, and results are shown in FIG. 7B (and Table 17B). Phosphoketolase strains appeared to have a decreased lag phase compared to controls, but the exponential growth rates were not statistically different (average rate of 0.16 h⁻¹).

TABLE 17A Condition 0 h 18.3 h Strain OD OD xpk ISO 1 0.1 2.3 xpk ISO 2 0.1 2.2 xpk ISO 3 0.1 2.2 ISO (no EtOH) 1 0.1 0.48 ISO (no EtOH) 2 0.1 0.41 ISO (no EtOH) 3 0.1 0.47 ISO (+EtOH) 1 0.1 2.5 ISO (+EtOH) 2 0.1 2.6 ISO (+EtOH) 3 0.1 2.4

TABLE 17B Condition 0 h 6.5 h 23 h 27 h 48 h Strain OD OD OD OD OD xpk ISO 1 0.1 0.18 2.91 4 4.4 xpk ISO 2 0.1 0.14 1.54 2.88 4.6 xpk ISO 3 0.1 0.16 2.15 3.54 4.3 ISO (+EtOH) 1 0.1 0.14 2.21 3.46 4.4 ISO (+EtOH) 2 0.1 0.11 1.13 2.18 4.2 ISO (+EtOH) 3 0.1 0.1 0.84 1.6 4.4

Example 10 Construction of Saccharomyces cerevisiae Strain BP913

The purpose of this example is to describe the construction of Saccharomyces cerevisiae strain BP913. The strain was derived from CEN.PK 113-7D (CBS 8340; Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, Netherlands) and contains deletions of the following genes: URA3, HIS3, PDC1, PDC5, and PDC6.

Deletions, which completely removed the entire coding sequence, were created by homologous recombination with PCR fragments containing regions of homology upstream and downstream of the target gene and either a G418 resistance marker or URA3 gene for selection of transformants. The G418 resistance marker, flanked by loxP sites, was removed using Cre recombinase. The URA3 gene was removed by homologous recombination to create a scarless deletion.

In general, the PCR cassette for each scarless deletion was made by combining four fragments, A-B-U-C, by overlapping PCR. The PCR cassette contained a selectable/counter-selectable marker, URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene, along with the promoter (250 bp upstream of the URA3 gene) and terminator (150 bp downstream of the URA3 gene). Fragments A and C, each 500 bp long, corresponded to the 500 bp immediately upstream of the target gene (Fragment A) and the 3′ 500 bp of the target gene (Fragment C). Fragments A and C were used for integration of the cassette into the chromosome by homologous recombination. Fragment B (500 bp long) corresponded to the 500 bp immediately downstream of the target gene and was used for excision of the URA3 marker and Fragment C from the chromosome by homologous recombination, as a direct repeat of the sequence corresponding to Fragment B was created upon integration of the cassette into the chromosome. Using the PCR product ABUC cassette, the URA3 marker was first integrated into and then excised from the chromosome by homologous recombination. The initial integration deleted the gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp region of the gene was also deleted. For integration of genes using this method, the gene to be integrated was included in the PCR cassette between fragments A and B.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-loxP cassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 661). pLA54 contains the K. lactis TEF1 promoter and kanMX marker, and is flanked by loxP sites to allow recombination with Cre recombinase and removal of the marker. PCR was done using Phusion DNA polymerase and primers BK505 and BK506 (SEQ ID NOs:662 and 663). The URA3 portion of each primer was derived from the 5′ region upstream of the URA3 promoter and 3′ region downstream of the coding region such that integration of the loxP-kanMX-loxP marker resulted in replacement of the URA3 coding region. The PCR product was transformed into CEN.PK 113-7D using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YPD containing G418 (100 μg/ml) at 30 C. Transformants were screened to verify correct integration by PCR using primers LA468 and LA492 (SEQ ID NOs:664 and 665) and designated CEN.PK 113-7D Δura3::kanMX. HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen; Valencia, Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO: 666) and primer oBP453 (SEQ ID NO: 667), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplified with primer oBP454 (SEQ ID NO: 668), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 669), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment U. HIS3 Fragment U was amplified with primer oBP456 (SEQ ID NO: 670), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQ ID NO: 671), containing a 5′ tail with homology to the 5′ end of HIS3 Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO: 672), containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U, and primer oBP459 (SEQ ID NO: 673). PCR products were purified with a PCR Purification kit (Qiagen, Valencia, Calif.). HIS3 Fragment AB was created by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment B and amplifying with primers oBP452 (SEQ ID NO: 666) and oBP455 (SEQ ID NO: 669). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3 Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQ ID NO: 670) and oBP459 (SEQ ID NO: 673). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.). The HIS3 ABUC cassette was created by overlapping PCR by mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying with primers oBP452 (SEQ ID NO: 666) and oBP459 (SEQ ID NO: 673). The PCR product was purified with a PCR Purification kit (Qiagen, Valencia, Calif.).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformed with the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, Calif.). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30 C. Transformants with a his3 knockout were screened for by PCR with primers oBP460 (SEQ ID NO: 674) and oBP461 (SEQ ID NO: 671) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was selected as strain CEN.PK 113-7D Δura3::kanMX Δhis3::URA3. KanMX Marker Removal from the Δura3 Site and URA3 Marker Removal from the Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMX Δhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 715) using a Frozen-EZ Yeast Transformation II kit (Zymo Research, Orange, Calif.) and plating on synthetic complete medium lacking histidine and uracil supplemented with 2% glucose at 30 C. Transformants were grown in YP supplemented with 1% galactose at 30 C for ˜6 hours to induce the Cre recombinase and KanMX marker excision and plated onto YPD (2% glucose) plates at 30 C for recovery. An isolate was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. 5-FOA resistant isolates were grown in and plated on YPD for removal of the pRS423::PGAL1-cre plasmid. Isolates were checked for loss of the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid by assaying growth on YPD+G418 plates, synthetic complete medium lacking uracil plates, and synthetic complete medium lacking histidine plates. A correct isolate that was sensitive to G418 and auxotrophic for uracil and histidine was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 and designated as BP857. The deletions and marker removal were confirmed by PCR and sequencing with primers oBP450 (SEQ ID NO: 676) and oBP451 (SEQ ID NO: 677) for Δura3 and primers oBP460 (SEQ ID NO: 674) and oBP461 (SEQ ID NO: 675) for Δhis3 using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen, Valencia, Calif.).

PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletion were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs, Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO: 670) and primer oBP441 (SEQ ID NO: 679), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment B. PDC6 Fragment B was amplified with primer oBP442 (SEQ ID NO: 680), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment A, and primer oBP443 (SEQ ID NO: 681), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment U. PDC6 Fragment U was amplified with primer oBP444 (SEQ ID NO: 682), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment B, and primer oBP445 (SEQ ID NO: 683), containing a 5′ tail with homology to the 5′ end of PDC6 Fragment C. PDC6 Fragment C was amplified with primer oBP446 (SEQ ID NO: 684), containing a 5′ tail with homology to the 3′ end of PDC6 Fragment U, and primer oBP447 (SEQ ID NO: 685). PCR products were purified with a PCR Purification kit (Qiagen). PDC6 Fragment AB was created by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment B and amplifying with primers oBP440 (SEQ ID NO: 678) and oBP443 (SEQ ID NO: 681). PDC6 Fragment UC was created by overlapping PCR by mixing PDC6 Fragment U and PDC6 Fragment C and amplifying with primers oBP444 (SEQ ID NO: 682) and oBP447 (SEQ ID NO: 685). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC6 ABUC cassette was created by overlapping PCR by mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying with primers oBP440 (SEQ ID NO: 678) and oBP447 (SEQ ID NO: 685). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made and transformed with the PDC6 ABUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30 C. Transformants with a pdc6 knockout were screened for by PCR with primers oBP448 (SEQ ID NO: 686) and oBP449 (SEQ ID NO: 687) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion and marker removal were confirmed by PCR and sequencing with primers oBP448 (SEQ ID NO: 686) and oBP449 (SEQ ID NO: 687) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC6 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC6, oBP554 (SEQ ID NO: 688) and oBP555 (SEQ ID NO: 689). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 and designated as BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region from Streptococcus mutans ATCC #700610 (SEQ ID NO: 1886). The A fragment followed by the ilvD coding region from Streptococcus mutans for the PCR cassette for the PDC1 deletion-ilvDSm integration was amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and NYLA83 genomic DNA as template (construction of strain NYLA83 is described in U.S. Application Pub. No. 20110124060 A1, incorporated herein by reference), prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 Fragment A-ilvDSm was amplified with primer oBP513 (SEQ ID NO: 690) and primer oBP515 (SEQ ID NO: 691), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment B. The B, U, and C fragments for the PCR cassette for the PDC1 deletion-ilvDSm integration were amplified using Phusion High Fidelity PCR Master Mix (New England BioLabs) and CEN.PK 113-7D genomic DNA as template, prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). PDC1 Fragment B was amplified with primer oBP516 (SEQ ID NO: 692), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQ ID NO: 693), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO: 694), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B, and primer oBP519 (SEQ ID NO: 695), containing a 5′ tail with homology to the 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primer oBP520 (SEQ ID NO: 696), containing a 5′ tail with homology to the 3′ end of PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 697). PCR products were purified with a PCR Purification kit (Qiagen). PDC1 Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513 (SEQ ID NO: 690) and oBP517 (SEQ ID NO: 693). PDC1 Fragment UC was created by overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C and amplifying with primers oBP518 (SEQ ID NO: 694) and oBP521 (SEQ ID NO: 697). The resulting PCR products were purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC1 A-ilvDSm-BUC cassette was created by overlapping PCR by mixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying with primers oBP513 (SEQ ID NO: 690) and oBP521 (SEQ ID NO: 697). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made and transformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 2% glucose at 30 C. Transformants with a pdc1 knockout ilvDSm integration were screened for by PCR with primers oBP511 (SEQ ID NO: 698) and oBP512 (SEQ ID NO: 699) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC1 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC1, oBP550 (SEQ ID NO: 700) and oBP551 (SEQ ID NO: 701). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grown overnight in YPD and plated on synthetic complete medium containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion of PDC1, integration of ilvDSm, and marker removal were confirmed by PCR and sequencing with primers oBP511 (SEQ ID NO: 698) and oBP512 (SEQ ID NO: 699) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm and designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region from Achromobacter xylosoxidans. A segment of the PCR cassette for the PDC5 deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS (described in Example 2). The coding sequence of sadB (SEQ ID NO: 718) and PDC5 Fragment B were cloned into pUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCR cassette. The coding sequence of sadB was amplified using pLH468-sadB (SEQ ID NO: 716) as template with primer oBP530 (SEQ ID NO: 702), containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 703), containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B. PDC5 Fragment B was amplified from CEN.PK 113-7D genomic DNA with primer oBP532 (SEQ ID NO: 704), containing a 5′ tail with homology to the 3′ end of sadB, and primer oBP533 (SEQ ID NO: 705), containing a PmeI restriction site. PCR products were purified with a PCR Purification kit (Qiagen). sadB-PDC5 Fragment B was created by overlapping PCR by mixing the sadB and PDC5 Fragment B PCR products and amplifying with primers oBP530 (SEQ ID NO: 702) and oBP533 (SEQ ID NO: 705). The resulting PCR product was digested with AscI and PmeI and ligated with T4 DNA ligase into the corresponding sites of pUC19-URA3MCS after digestion with the appropriate enzymes. The resulting plasmid was used as a template for amplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ ID NO: 706) and oBP546 (SEQ ID NO: 707), containing a 5′ tail with homology to the 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplified from CEN.PK 113-7D genomic DNA with primer oBP547 (SEQ ID NO: 708), containing a 5′ tail with homology to the 3′ end of PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 709). PCR products were purified with a PCR Purification kit (Qiagen). PDC5 sadB-Fragment B-Fragment U-Fragment C was created by overlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U and PDC5 Fragment C and amplifying with primers oBP536 (SEQ ID NO: 706) and oBP539 (SEQ ID NO: 709). The resulting PCR product was purified on an agarose gel followed by a Gel Extraction kit (Qiagen). The PDC5 A-sadB-BUC cassette was created by amplifying PDC5 sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ ID NO: 710) containing a 5′ tail with homology to the 50 nucleotides immediately upstream of the native PDC5 coding sequence, and oBP539 (SEQ ID NO: 709). The PCR product was purified with a PCR Purification kit (Qiagen).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm were made and transformed with the PDC5 A-sadB-BUC PCR cassette using a Frozen-EZ Yeast Transformation II kit (Zymo Research). Transformation mixtures were plated on synthetic complete media lacking uracil supplemented with 1% ethanol (no glucose) at 30 C. Transformants with a pdc5 knockout sadB integration were screened for by PCR with primers oBP540 (SEQ ID NO: 711) and oBP541 (SEQ ID NO: 712) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The absence of the PDC5 gene from the isolate was demonstrated by a negative PCR result using primers specific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 713) and oBP553 (SEQ ID NO: 714). A correct transformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 was grown overnight in YPE (1% ethanol) and plated on synthetic complete medium supplemented with ethanol (no glucose) and containing 5-fluoro-orotic acid (0.1%) at 30 C to select for isolates that lost the URA3 marker. The deletion of PDC5, integration of sadB, and marker removal were confirmed by PCR with primers oBP540 (SEQ ID NO: 711) and oBP541 (SEQ ID NO: 712) using genomic DNA prepared with a Gentra Puregene Yeast/Bact kit (Qiagen). The correct isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB and designated as BP913.

Example 11 Construction of Strain NYLA83

This example describes insertion-inactivation of endogenous PDC1 and PDC6 genes of S. cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase. The resulting strain was used as described in Example 10.

Construction of pRS425::GPM-sadB

A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 717) from Achromobacter xylosoxidans (disclosed in US Patent Application Publication No. US20090269823) was cloned. The coding region of this gene called sadB for secondary alcohol dehydrogenase (SEQ ID NO: 718) was amplified using standard conditions from A. xylosoxidans genomic DNA, prepared using a Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5500A) following the recommended protocol for gram negative organisms using forward and reverse primers N473 and N469 (SEQ ID NOs:725 and 726), respectively. The PCR product was TOPO-Blunt cloned into pCR4 BLUNT (Invitrogen) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-I cells. Plasmid was subsequently isolated from four clones, and the sequence verified.

The sadB coding region was PCR amplified from pCR4Blunt::sadB. PCR primers contained additional 5′ sequences that would overlap with the yeast GPM1 promoter and the ADH1 terminator (N583 and N584, provided as SEQ ID NOs:727 and 728). The PCR product was then cloned using “gap repair” methodology in Saccharomyces cerevisiae (Ma et al. ibid) as follows. The yeast-E. coli shuttle vector pRS425::GPM::kivD::ADH which contains the GPM1 promoter (SEQ ID NO:721), kivD coding region from Lactococcus lactis (SEQ D NO:719), and ADH1 terminator (SEQ ID NO:722) (described in U.S. Pat. No. 7,851,188, Example 17) was digested with BbvCI and PacI restriction enzymes to release the kivD coding region. Approximately 1 μg of the remaining vector fragment was transformed into S. cerevisiae strain BY4741 along with 1 μg of sadB PCR product. Transformants were selected on synthetic complete medium lacking leucine. The proper recombination event, generating pRS425::GPM-sadB, was confirmed by PCR using primers N142 and N459 (SEQ ID NOs:729 and 730).

Construction of pdc6:: P_(GPM1)-sadB Integration Cassette and PDC6 Deletion:

A pdc6::P_(GPM1)-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO:723) from pRS425::GPM-sadB (SEQ ID NO: 720) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:724) contains the URA3 marker from pRS426 (ATCC #77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 114117-11A through 114117-11D (SEQ ID NOs:731, 732, 733 and 734), and 114117-13A and 114117-13B (SEQ ID NOs:735 and 736).

The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′˜50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC #200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs:737 and 738), and 112590-34F and 112590-49E (SEQ ID NOs: 739 and 740) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::P_(GPM1)-sadB-ADH1t.

Construction of pdc1::P_(PDC1)-ilvD Integration Cassette and PDC1 Deletion:

A pdc1::P_(PDC1)-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO:741) from pLH468 (SEQ ID NO: 1888) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-540S) and primers 114117-27A through 114117-27D (SEQ ID NOs:742, 743, 744 and 745).

The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′˜50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::P_(GPM1)-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs 746 and 747), and primers 112590-49E and 112590-30F (SEQ ID NOs 740 and 748) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6.: P_(GPM1)-sadB-ADHt pdc1::_P_(PDC1)-ilvD-FBA1t.

HIS3 Deletion

To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO: 749). URA3r2 contains the URA3 marker from pRS426 (ATCC #77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion DNA polymerase and primers 114117-45A and 114117-45B (SEQ ID NOs:750 and 751) which generated a ˜2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain, called NYLA73, has the genotype: BY4700 pdc6:: P_(GPM1)-sadB-ADH1t pdc1:: P_(PDC1)-ilvD-FBA1t Δhis3.

Construction of Pdc5::kanMX Integration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 4034091) using Phusion DNA polymerase and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs:752 and 753) which generated a ˜2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 754 and 755). The identified correct transformants have the genotype: BY4700 pdc6:: P_(GPM1)-sadB-ADH1t pdc1:: P_(PDC1)-ilvD-FBA1t Δhis3 pdc5::kanMX4. The strain was named NYLA74.

Deletion of HXK2 (Hexokinase II):

A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (described above) using Phusion DNA polymerase and primers 384 and 385 (SEQ ID NOs:756 and 757) which generated a ˜2.3 kb PCR product. The HXK2 portion of each primer was derived from the 5′ region upstream of the HXK2 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HXK2 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened by PCR to verify correct integration at the HXK2 locus with replacement of the HXK2 coding region using primers N869 and N871 (SEQ ID NOs:758 and 759). The URA3r2 marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth, and by PCR to verify correct marker removal using primers N946 and N947 (SEQ ID NOs:760 and 761). The resulting identified strain named NYLA83 has the genotype: BY4700 pdc6.: P_(GPM1)-sadB-ADH1t pdc1:: P_(PDC1)-ilvD-FBA1t Δhis3 Δhxk2.

Example 12 Construction of Saccharomyces cerevisiae Strain PNY2257

Strain PNY2242 was constructed in several steps from BP913 (described above). First, the native GPD2 gene on Chromosome XV was deleted. The coding region was deleted using CRE-lox mediated marker removal (methodology described above), so the resulting locus contains one loxP site. The sequence of the modified locus is provided as SEQ ID NO: 1899 (Upstream region=nt 1-500; loxP site=nt 531-564; Downstream region=nt 616-1115). Second, the native FRA2 gene on Chromosome VII was deleted. Elimination of FRA2 was a scarless deletion of only the coding region. The sequence of the modified locus is provided as SEQ ID NO: 1900 (Upstream region=nt 1-501; Downstream region=nt 526-1025). Next, the ADH1 gene on Chromosome XV was deleted along with insertion of a chimeric gene comprised of the UAS(PGK1)-FBA1 promoter and the kivD coding region. The native ADH1 terminator was used to complete the gene. The sequence of the modified locus is provided as SEQ ID No. 1901 (Upstream region=nt 1-500; UAS(PGK1)FBA promoter=nt 509-1233; kivD coding region=nt 1242-2888; Downstream region (includes terminator)=nt 2889-3388). Next, a chimeric gene comprised of the FBA1 promoter, the alsS coding region and the CYC1 terminator was integrated into Chromosome XII, upstream of the TRX1 gene. The sequence of the modified locus is provided as SEQ ID No. 1902 (Upstream region=nt 1-154; FBA1 promoter=nt 155-802; alsS CDS=nt 810-2525; CYC1 terminator=nt 2534-2788; Downstream region=nt 2790-3015). Next, two copies of a gene encoding horse liver alcohol dehydrogenase were integrated into Chromosomes VII and XVI. On Chromosome VII, a chimeric gene comprised of the PDC1 promoter, the hADH coding region and the ADH1 terminator were placed into the fra2Δ locus (the original deletion of FRA2 is described above). The sequence of the modified locus is provided as SEQ ID No. 1903 (Upstream region=nt 1-300; PDC1 promoter=nt 309-1178; hADH coding region=nt 1179-2306; ADH1 terminator=nt 2315-2630; Downstream region=nt 2639-2900). On Chromosome XVI, a chimeric gene comprised of the PDC5 promoter, the hADH coding region and the ADH1 terminator were integrated in the region formerly occupied by the long term repeat element YPRCdelta15. The sequence of the modified locus is provided as SEQ ID No. 1904 (Upstream region=nt 1-150; PDC5 promoter=nt 159-696; hADH coding region=nt 697-1824; ADH1 terminator=nt 1833-2148; Downstream region=nt 2157-2656). Then the native genes YMR226c and ALD6 were deleted. Elimination of YMR226c was a scarless deletion of only the coding region. The sequence of the modified locus is provided as SEQ ID No. 1905 (Upstream region=nt 1-250; Downstream region=nt 251-451). The ALD6 coding region plus 700 bp of upstream sequence were deleted using CRE-lox mediated marker removal, so the resulting locus contains one loxP site. The sequence of the modified locus is provided as SEQ ID No. 1906 (Upstream region=nt 1-500; loxP site=nt 551-584; Downstream region=nt 678-1128). The geneticin-selectable phosphoketolase expression vector described in Example 8 was transformed into the strain and confirmed as described above (the locus is depicted in FIG. 6). Finally, plasmids were introduced into the strain for expression of KARI (pLH702, plasmid SEQ ID. No. 1907) and DHAD (pYZ067DkivDDhADH, SEQ ID. No. 1908), resulting in the strain named PNY2257. A control strain containing all of the elements above except for the phosphoketolase pathway construct is called PNY2242.

Growth rates were assessed as described in previous examples. Over a 24 hour period, PNY2257 displayed growth rates without ethanol or other two-carbon supplement similar to those growth rates observed for PNY2242 with supplementation. 

What is claimed is:
 1. A method for the production of isobutanol comprising growing a recombinant host cell comprising: (i) at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity; (ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity, wherein the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is xpk1 from Lactobacillus plantarum, xpkA from Lactobacillus pentosus MD363, or 6-phosphate phosphoketolase from B. lactis; (iii) at least one genetic modification in an endogenous gene encoding a polypeptide having glycerol-3-phosphate dehydrogenase activity, wherein the at least one genetic modification reduces or eliminates glycerol-3-phosphate dehydrogenase activity; and (iv) an isobutanol biosynthetic pathway comprising the substrate to product conversions: (a) pyruvate to acetolactate, (b) acetolactate to 2,3-dihydroxyisovalerate, (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate, (d) 2-ketoisovalerate to isobutyraldehyde, (e) isobutyraldehyde to isobutanol; under conditions wherein isobutanol is produced and optionally recovering isobutanol.
 2. The method of claim 1, wherein the isobutanol is recovered by distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, pervaporation, vacuum flash, or combinations thereof.
 3. The method of claim 1, wherein the isobutanol is recovered by contacting the fermentation broth with an extractant.
 4. The method of claim 3, wherein the extractant is C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, or mixtures thereof.
 5. The recombinant host cell of claim 1, wherein the polypeptide having phosphoketolase activity comprises at least 85% identity to SEQ ID NO:
 481. 6. The recombinant host cell of claim 1, wherein the recombinant host cell is Saccharomyces cerevisiae. 